Extraction solvents derived from oil for alcohol removal in extractive fermentation

ABSTRACT

In an alcohol fermentation process, oil derived from biomass is hydrolyzed into an extractant available for in situ removal of a product alcohol such as butanol from a fermentation broth. The glycerides in the oil can be catalytically (e.g., enzymatically) hydrolyzed into free fatty acids, which form a fermentation product extractant having a partition coefficient for a product alcohol greater than a partition coefficient of the oil of the biomass for the product alcohol. Oil derived from a feedstock of an alcohol fermentation process can be hydrolyzed by contacting the feedstock including the oil with one or more enzymes whereby at least a portion of the oil is hydrolyzed into free fatty acids forming a fermentation product extractant, or the oil can be separated from the feedstock prior to the feedstock being fed to a fermentation vessel, and the separated oil can be contacted with the enzymes to form the fermentation product extractant. The fermentation product extractant can be contacted with a fermentation broth for in situ removal of a product alcohol.

This application claims the benefit of U.S. Provisional Application No.61/356,290, filed on Jun. 18, 2010; U.S. Provisional Application No.61/368,451, filed on Jul. 28, 2010; U.S. Provisional Application No.61/368,436, filed on Jul. 28, 2010; U.S. Provisional Application No.61/368,444, filed on Jul. 28, 2010; U.S. Provisional Application No.61/368,429, filed on Jul. 28, 2010; U.S. Provisional Application No.61/379,546, filed on Sep. 2, 2010; and U.S. Provisional Application No.61/440,034, filed on Feb. 7, 2011; U.S. patent application Ser. No.13/160,766, filed on Jun. 15, 2011; the entire contents of which are allherein incorporated by reference.

The Sequence Listing associated with this application is filed inelectronic form via EFS-Web and hereby incorporated by reference intothe specification in its entirety.

FIELD OF THE INVENTION

The present invention relates the production of fermentative alcoholssuch as butanol, and in particular to extraction solvents for extractivefermentation and processes for converting oil derived from biomass intothe extraction solvents.

BACKGROUND OF THE INVENTION

Alcohols have a variety of applications in industry and science such asa beverage (i.e., ethanol), fuel, reagents, solvents, and antiseptics.For example, butanol is an alcohol that is an important industrialchemical with a variety of applications including use as a fueladditive, as a feedstock chemical in the plastics industry, and as afood-grade extractant in the food and flavor industry. Accordingly,there is a high demand for alcohols such as butanol, as well as forefficient and environmentally-friendly production methods.

Production of alcohol utilizing fermentation by microorganisms is onesuch environmentally-friendly production method. In the production ofbutanol, in particular, some microorganisms that produce butanol in highyields also have low butanol toxicity thresholds. Removal of butanolfrom the fermentation vessel as it is being produced is a means tomanage these low butanol toxicity thresholds.

In situ product removal (ISPR) (also referred to as extractivefermentation) can be used to remove butanol (or other fermentativealcohol) from the fermentation vessel as it is produced, therebyallowing the microorganism to produce butanol at high yields. One ISPRmethod for removing fermentative alcohol that has been described in theart is liquid-liquid extraction (U.S. Patent Application Publication No.2009/0305370). In order to be technically and economically viable,liquid-liquid extraction calls for good contact between the extractantand the fermentation broth for efficient mass transfer of the productalcohol into the extractant; good phase separation of the extractantfrom the fermentation broth (during and/or after fermentation);efficient recovery and recycle of the extractant; minimal degradation ofthe ability of the extractant to extract the product alcohol (e.g., bypreventing the lowering of the partition coefficient for the productalcohol into the extractant); and minimal contamination of theextractant by lipids that lower the partition coefficient over along-term operation.

The partition coefficient of the extractant can be degraded over timewith each recycle, for example, by the build-up of lipids present in thebiomass that is fed to the fermentation vessel as feedstock ofhydrolysable starch. As an example, a liquefied corn mash loaded to afermentation vessel at 30 wt % dry corn solids can result in afermentation broth that contains about 1.2 wt % corn oil duringconversion of glucose to butanol by simultaneous saccharification andfermentation (SSF) (with saccharification of the liquefied mashoccurring during fermentation by the addition of glucoamylase to produceglucose). The dissolution of the corn oil lipids in oleyl alcohol (OA)serving as an extractant during ISPR can result in build-up of lipidconcentration with each OA recycle, decreasing the partition coefficientfor the product alcohol in OA as the lipid concentration in OA increaseswith each recycle of OA.

In addition, the presence of the undissolved solids during extractivefermentation can negatively affect the efficiency of alcohol production.For example, the presence of the undissolved solids may lower the masstransfer coefficient inside the fermentation vessel, impede phaseseparation in the fermentation vessel, result in the accumulation ofcorn oil from the undissolved solids in the extractant leading toreduced extraction efficiency over time, increase the loss of solventbecause it becomes trapped in solids and ultimately removed as DriedDistillers' Grains with Solubles (DDGS), slow the disengagement ofextractant drops from the fermentation broth, and/or result in a lowerfermentation vessel volume efficiency.

Several approaches for reducing the degradation of the extractant usedin extractive fermentation with lipid have included biomass wet milling,fractionation, and removal of solids. Wet milling is an expensive,multi-step process that separates a biomass (e.g., corn) into its keycomponents (germ, pericarp fiber, starch, and gluten) in order tocapture value from each co-product separately. This process gives apurified starch stream; however, it is costly and includes theseparation of the biomass into its non-starch components which isunnecessary for fermentative alcohol production. Fractionation removesfiber and germ which contains a majority of the lipids present in groundwhole grain such as corn, resulting in corn that has a higher starch(endosperm) content. Dry fractionation does not separate the germ andfiber and therefore, it is less expensive than wet milling. However,fractionation does not remove the entirety of the fiber or germ, anddoes not result in total elimination of solids. Furthermore, there issome loss of starch in fractionation. Wet milling of corn is moreexpensive than dry fractionation, but dry fractionation is moreexpensive than dry grinding of unfractionated corn. Removal of solidsincluding germ containing lipids, from liquefied mash prior to use infermentation can substantially eliminate undissolved solids asdescribed, for example, in co-pending, commonly owned U.S. ProvisionalApplication Ser. No. 61/356,290, filed Jun. 18, 2010. However, it wouldbe advantageous if the degradation of the partition coefficient of theextractant can be reduced even without fractionation or removal ofundissolved solids. Thus, there is a continuing need to develop moreefficient methods and systems for producing product alcohols, such asbutanol, through extractive fermentation in which the degradation of thepartition coefficient of the extractant is reduced.

Moreover, the extractant (e.g., oleyl alcohol) is typically added to thefermentation process, rather than produced at a step in the process andtherefore, the extractant is a raw material expense. Since extractantcan be lost by adsorption on non-fermentable solids and/or diluted bylipids introduced into the fermentation process, the economics of analcohol production process can be affected by the efficiency of theextractant recovery and recycle. Thus, there exists a continuing needfor alternative extractants for ISPR that can result in a moreeconomical process by reducing capital and/or operating costs.

BRIEF SUMMARY OF THE INVENTION

The present invention satisfies the above needs by providing methods forproducing product alcohols such as butanol, in which the lipids in abiomass are converted into an extractant that can be used in ISPR, andin which the amount of lipids that are fed to the fermentation vesselwith the feedstock and/or upon extractant recycle, are decreased. Thepresent invention offers a solution to the degradation of the ability ofthe extractant to extract a product alcohol (e.g., butanol) bypreventing the lowering of the partition coefficient for the productalcohol into the extractant. The application offers a solution to thecontamination of the extractant by triglycerides that lower thepartition coefficient of the extractant for a product alcohol. Thepresent invention provides further related advantages as will be madeapparent by the description of the embodiments that follow.

Catalytic (e.g., enzymatic) hydrolysis of lipids derived from biomassinto fatty acids can decrease the rate of undesirable build-up of lipidsin the ISPR extractant. The fatty acids can be obtained from hydrolysisof lipids found in the biomass which supplies the fermentable carbon forfermentation. Fatty acids would not be expected to decrease thepartition coefficient of the product alcohol such as a butanol into theextractant phase as much as the lipids, as the partition coefficient forbutanol from water to fatty acids has been determined to besignificantly greater than the partition coefficient for butanol fromwater to fatty acid esters or triglycerides. Moreover, the fatty acidscan be used as an ISPR extractant which can be produced at a step in thealcohol production process and can be used in place of, or in additionto, a supplied, exogenous ISPR extractant that is not produced in theprocess (such as, but not limited to, oleyl alcohol or oleic acid),thereby reducing the raw material expense for the ISPR extractant.

In one embodiment, the present invention is directed to a methodcomprising contacting biomass comprising water, fermentable carbonsource, and oil with one or more catalyst whereby at least a portion ofthe oil is hydrolyzed by one or more catalyst to form an extractant,wherein the fermentable carbon source and the oil are both derived fromthe biomass. The biomass may comprises corn grain, corn cobs, cropresidues, corn husks, corn stover, grasses, wheat, rye, wheat straw,barley, barley straw, hay, rice straw, switchgrass, waste paper, sugarcane bagasse, sorghum, sugar cane, soy, grains, cellulosic material,lignocellulosic material, trees, branches, roots, leaves, wood chips,sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure,and mixtures thereof. In a further embodiment, the oil may compriseglycerides and one or more catalysts may hydrolyze the glycerides toform fatty acids. In another embodiment, the one or more catalysts maybe selected from esterase, lipase, phospholipase, and lysophospholipase.

In another embodiment, the extractant may comprise fatty acids, fattyamides, fatty alcohols, fatty esters, triglycerides, or mixturesthereof. In a further embodiment, the extractant may comprise a mixtureof fatty acids or a mixture of fatty acids and fatty amides. In afurther embodiment, a partition coefficient of the extractant for theproduct alcohol may be greater than a partition coefficient of the oilof the biomass for the product alcohol.

The method of the present invention may further comprise the step ofinactivating the catalyst after at least a portion of the oil ishydrolyzed. In another embodiment, the method may further comprise thestep of separating the oil from the biomass prior to hydrolysis by oneor more catalyst. The claimed method may also further comprise the stepsof contacting the biomass with a fermentation broth in a fermentationvessel; fermenting the carbon source of the biomass to produce a productalcohol; and removing in situ the product alcohol from the fermentationbroth by contacting the broth with the extractant. The product alcoholmay be butanol.

In another embodiment, the present invention is directed to a method forproducing an alcohol comprising (a) providing biomass comprising water,fermentable carbon source, and oil; (b) liquefying the biomass toproduce a liquefied biomass; (c) contacting the liquefied biomass withone or more catalysts whereby at least a portion of the oil ishydrolyzed to form an extractant; (d) contacting the liquefied biomasswith a saccharification enzyme capable of converting oligosaccharidesinto fermentable sugar; (e) contacting the liquefied biomass with afermentation broth in a fermentation vessel; (f) fermenting the carbonsource of the liquefied biomass to produce a product alcohol; (g)removing in situ the product alcohol from the fermentation broth bycontacting the broth with the extractant; and optionally steps (c) and(d) occur concurrently. The biomass may comprises corn grain, corn cobs,crop residues, corn husks, corn stover, grasses, wheat, rye, wheatstraw, barley, barley straw, hay, rice straw, switchgrass, waste paper,sugar cane bagasse, sorghum, sugar cane, soy, grains, cellulosicmaterial, lignocellulosic material, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animalmanure, and mixtures thereof. In a further embodiment, the oil maycomprise glycerides and one or more catalysts may hydrolyze theglycerides to form fatty acids. In another embodiment, the one or morecatalysts may be selected from esterase, lipase, phospholipase, andlysophospholipase. In another embodiment, the extractant may comprisefatty acids, fatty amides, fatty alcohols, fatty esters, triglycerides,or mixtures thereof. In a further embodiment, the extractant maycomprise a mixture of fatty acids or a mixture of fatty acids or fattyamides. In a further embodiment, a partition coefficient of theextractant for the product alcohol may be greater than a partitioncoefficient of the oil of the biomass for the product alcohol. Themethod of the present invention may further comprise the step ofinactivating the catalyst after at least a portion of the oil ishydrolyzed. The product alcohol may be butanol.

The present invention is also directed to a composition comprising arecombinant microorganism capable of producing an alcohol; fermentablecarbon source; one or more catalysts capable of hydrolyzing glyceridesinto fatty acids; oil comprising glycerides; and fatty acids. The one ormore catalysts may be selected from esterase, lipase, phospholipase, andlysophospholipase, and the oil may be corn, tallow, canola,capric/caprylic triglycerides, castor, coconut, cottonseed, fish,jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed,rice, safflower, soya, sunflower, tung, jatropha and vegetable oilblends. In a further embodiment, the fermentable carbon source and theoil are derived from biomass. The biomass may comprise corn grain, corncobs, crop residues, corn husks, corn stover, grasses, wheat, rye, wheatstraw, barley, barley straw, hay, rice straw, switchgrass, waste paper,sugar cane bagasse, sorghum, sugar cane, soy, grains, cellulosicmaterial, lignocellulosic material, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animalmanure, and mixtures thereof. The composition may further comprise asaccharification enzyme and/or undissolved solids. The composition mayalso comprise at least one or more of monoglycerides, diglycerides,triglycerides, glycerol, monosaccharides, oligosaccharides, or alcohol.In addition, the alcohol may be butanol.

In some embodiments, a method of removing oil derived from biomass froma fermentation process includes contacting an aqueous biomass feedstreamwith a catalyst. The feedstream includes water, fermentable carbon andan amount of oil, and the fermentable carbon and the oil are bothderived from the biomass. At least a portion of the oil is hydrolyzedaccording to methods described in the present invention into fatty acidsto form a catalyst-treated biomass feedstream including the fatty acids.

In some embodiments, a method of producing an extractant for in situremoval of a product alcohol includes providing biomass which includessugar and oil, the oil having an amount of triglycerides, and contactingthe oil with a composition including one or more enzymes capable ofhydrolyzing the triglycerides into fatty acids. The triglycerides in theoil are hydrolyzed to form a fermentation product extractant having apartition coefficient for the product alcohol greater than a partitioncoefficient of the oil of the biomass for the product alcohol.

In some embodiments, a method for producing butanol includes (a)providing biomass having starch and oil, the oil including an amount ofglycerides; (b) liquefying the biomass to produce a liquefied biomass,the liquefied biomass including oligosaccharides hydrolyzed from thestarch; (c) contacting the biomass of step (a) or the liquefied biomassof step (b) with a composition having one or more enzymes capable ofconverting the glycerides into free fatty acids whereby the free fattyacids form a fermentation product extractant; (d) contacting theliquefied biomass with a saccharification enzyme capable of convertingoligosaccharides into fermentable sugar including monomeric glucose; (e)contacting the liquefied biomass with a biocatalyst capable ofconverting the fermentable sugar to butanol whereby a fermentationproduct comprising butanol is produced; and (f) contacting thefermentation product with the fermentation product extractant wherebythe butanol is separated from the fermentation product, the fermentationproduct extractant having a partition coefficient for the butanolgreater than a partition coefficient of the oil of the biomass for thebutanol.

In some embodiments, a method includes, at a step during a process toproduce a product alcohol from a feedstock, contacting the productalcohol with an extractant comprising free fatty acids obtained fromenzymatic hydrolysis of a native oil wherein the oil comprisesglycerides. The extractant has a partition coefficient for the productalcohol greater than a partition coefficient of the native oil for theproduct alcohol.

In some embodiments, the process to produce a product alcohol from afeedstock includes (a) liquefying the feedstock to create a feedstockslurry; (b) centrifuging the feedstock slurry of (a) to produce acentrifuge product including (i) an aqueous layer comprising sugar, (ii)a plant-derived oil layer, and (iii) a solids layer; (c) feeding theaqueous layer of (b) to a fermentation vessel; and (d) fermenting thesugar of the aqueous layer to produce the product alcohol.

In some embodiments, the process to produce a product alcohol from afeedstock further includes adding the extractant to the fermentationvessel to form a two-phase mixture comprising an aqueous phase and aproduct alcohol-containing organic phase.

In some embodiments, the native oil is a plant-derived oil, and in someembodiments, the process to produce a product alcohol from a feedstockfurther includes obtaining the plant-derived oil from the plant-derivedoil layer; and converting the plant-derived oil into the extractant bycontacting the oil with one or more enzymes that hydrolyze theglycerides into free fatty acids.

In some embodiments, the process to produce a product alcohol from afeedstock further includes inactivating the one or more enzymes after atleast a portion of the glycerides have been hydrolyzed into free fattyacids.

In some embodiments, the process to produce a product alcohol from afeedstock further includes feeding the plant-derived oil to thefermentation vessel prior to the step of converting the plant-derivedoil into the extractant.

In some embodiments, the process to produce a product alcohol from afeedstock further includes adding a second extractant to thefermentation vessel to form a two-phase mixture comprising an aqueousphase and a product alcohol-containing organic phase.

In some embodiments, the plant-derived oil is converted to theextractant after the step of adding a second extractant.

In some embodiments, a method of removing oil derived from biomass froma fermentation process, includes (a) providing a fermentation brothcomprising a product alcohol and oil derived from biomass, the oilincluding glycerides; (b) contacting the fermentation broth with a firstextractant to form a two-phase mixture comprising an aqueous phase andan organic phase, wherein the product alcohol and the oil partition intothe organic phase to form a product alcohol-containing organic phase;(c) separating the product alcohol-containing organic phase from theaqueous phase; (d) separating the product alcohol from the organic phaseto produce a lean organic phase; and (e) contacting the lean organicphase with a composition comprising one or more catalysts capable ofhydrolyzing the glycerides into free fatty acids to produce a secondextractant comprising at least a portion of the first extractant andfree fatty acids.

In some embodiments, the method further includes repeating step (b) bycontacting the fermentation broth with the second extractant of step(e).

In some embodiments, an in situ fermentation extractant-formingcomposition includes (a) mash formed from biomass and including water,starch and oil, (b) a catalyst capable of hydrolyzing at least a portionof the triglycerides into free fatty acids, and (c) free fatty acids.The starch and the oil are both derived from the biomass, and the oilincludes an amount of triglycerides.

In some embodiments, a fermentation broth includes (a) a recombinantmicroorganism capable of producing butanol, (b) oligosaccharides, (c) acatalyst for hydrolyzing glycerides into free fatty acids, (d)glycerides, and (e) free fatty acids.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

The accompanying drawings, which are incorporated herein and form a partof the specification, illustrate the present invention and, togetherwith the description, further serve to explain the principles of theinvention and to enable a person skilled in the pertinent art to makeand use the invention.

FIG. 1 schematically illustrates an exemplary method and system of thepresent invention, in which a liquefied biomass is contacted with acatalyst for lipid hydrolysis before fermentation.

FIG. 2 schematically illustrates an exemplary method and system of thepresent invention, in which a liquefied and saccharified biomass iscontacted with a catalyst for lipid hydrolysis before fermentation.

FIG. 3 schematically illustrates an exemplary method and system of thepresent invention, in which lipids in a biomass feedstream are contactedwith a catalyst for lipid hydrolysis before or during liquefaction.

FIG. 4 schematically illustrates an exemplary method and system of thepresent invention, in which undissolved solids and lipids are removedfrom a liquefied biomass before fermentation, and in which the removedlipids are hydrolyzed into free fatty acids using a catalyst, and thefree fatty acids are supplied to the fermentation vessel.

FIG. 5 schematically illustrates an exemplary method and system of thepresent invention, in which lipids derived from native oil arehydrolyzed into free fatty acids using a catalyst, and the free fattyacids are supplied to the fermentation vessel.

FIG. 6 schematically illustrates an exemplary method and system of thepresent invention, in which biomass lipids present in a first extractantexiting a fermentation vessel are converted into free fatty acids thatare supplied to a fermentation vessel as a second extractant.

FIG. 7 is a chart illustrating the effect that the presence of fattyacids in a fermentation vessel has on glucose consumption forbutanologen strain NGCI-047.

FIG. 8 is a chart illustrating the effect that the presence of fattyacids in a fermentation vessel has on glucose consumption forbutanologen strain NGCI-049.

FIG. 9 is a chart illustrating the effect that the presence of fattyacids in a fermentation vessel has on glucose consumption forbutanologen strain NYLA84.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes.

In order to further define this invention, the following terms anddefinitions are herein provided.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains,” or “containing,” or any othervariation thereof, will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers. For example, a composition, a mixture, a process,a method, an article, or an apparatus that comprises a list of elementsis not necessarily limited to only those elements but can include otherelements not expressly listed or inherent to such composition, mixture,process, method, article, or apparatus. Further, unless expressly statedto the contrary, “or” refers to an inclusive or and not to an exclusiveor. For example, a condition A or B is satisfied by any one of thefollowing: A is true (or present) and B is false (or not present), A isfalse (or not present) and B is true (or present), and both A and B aretrue (or present).

Also, the indefinite articles “a” and “an” preceding an element orcomponent of the invention are intended to be nonrestrictive regardingthe number of instances, that is, occurrences of the element orcomponent. Therefore “a” or “an” should be read to include one or atleast one, and the singular word form of the element or component alsoincludes the plural unless the number is obviously meant to be singular.

The term “invention” or “present invention” as used herein is anon-limiting term and is not intended to refer to any single embodimentof the particular invention but encompasses all possible embodiments asdescribed in the application.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates orsolutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or to carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about,” the claims include equivalents to the quantities. Inone embodiment, the term “about” means within 10% of the reportednumerical value, alternatively within 5% of the reported numericalvalue.

“Biomass” as used herein refers to a natural product containinghydrolyzable polysaccharides that provide fermentable sugars includingany sugars and starch derived from natural resources such as corn, cane,wheat, cellulosic or lignocellulosic material and materials comprisingcellulose, hemicellulose, lignin, starch, oligosaccharides,disaccharides and/or monosaccharides, and mixtures thereof. Biomass mayalso comprise additional components such as protein and/or lipids.Biomass may be derived from a single source or biomass can comprise amixture derived from more than one source. For example, biomass maycomprise a mixture of corn cobs and corn stover, or a mixture of grassand leaves. Biomass includes, but is not limited to, bioenergy crops,agricultural residues, municipal solid waste, industrial solid waste,sludge from paper manufacture, yard waste, wood and forestry waste.Examples of biomass include, but are not limited to, corn grain, corncobs, crop residues such as corn husks, corn stover, grasses, wheat,rye, wheat straw, barley, barley straw, hay, rice straw, switchgrass,waste paper, sugar cane bagasse, sorghum, sugar cane, soy, componentsobtained from milling of grains, trees, branches, roots, leaves, woodchips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animalmanure, and mixtures thereof. For example, mash, juice, molasses, orhydrolysate may be formed from biomass by any processing known in theart for processing the biomass for purposes of fermentation such as bymilling, treating, and/or liquefying and comprises fermentable sugar andmay comprise water. For example, cellulosic and/or lignocellulosicbiomass may be processed to obtain a hydrolysate containing fermentablesugars by any method known to one skilled in the art. A low ammoniapretreatment is disclosed in U.S. Patent Application Publication No.2007/0031918A1, which is herein incorporated by reference. Enzymaticsaccharification of cellulosic and/or lignocellulosic biomass typicallymakes use of an enzyme consortium for breaking down cellulose andhemicellulose to produce a hydrolysate containing sugars includingglucose, xylose, and arabinose. (Saccharification enzymes suitable forcellulosic and/or lignocellulosic biomass are reviewed in Lynd, et al.(Microbiol. Mol. Biol. Rev. 66:506-577, 2002).

Mash, juice, molasses, or hydrolysate may include feedstock 12 andfeedstock slurry 16 as described herein. An aqueous feedstream may bederived or formed from biomass by any processing known in the art forprocessing the biomass for purposes of fermentation such as by milling,treating, and/or liquefying and comprises fermentable carbon substrate(e.g., sugar) and may comprise water. An aqueous feedstream may includefeedstock 12 and feedstock slurry 16 as described herein.

“Feedstock” as used herein means a feed in a fermentation process, thefeed containing a fermentable carbon source with or without undissolvedsolids, and where applicable, the feed containing the fermentable carbonsource before or after the fermentable carbon source has been liberatedfrom starch or obtained from the break down of complex sugars by furtherprocessing such as by liquefaction, saccharification, or other process.Feedstock includes or is derived from a biomass. Suitable feedstocksinclude, but are not limited to, rye, wheat, corn, cane, barley,cellulosic material, lignocellulosic material, or mixtures thereof.

“Fermentation broth” as used herein means the mixture of water, sugars,dissolved solids, optionally microorganisms producing alcohol, productalcohol, and all other constituents of the material held in thefermentation vessel in which product alcohol is being made by thereaction of sugars to alcohol, water, and carbon dioxide (CO₂) by themicroorganisms present. From time to time, as used herein the term“fermentation medium” and “fermented mixture” can be used synonymouslywith “fermentation broth.”

“Fermentable carbon source” or “fermentable carbon substrate” as usedherein means a carbon source capable of being metabolized by themicroorganisms disclosed herein for the production of fermentativealcohol. Suitable fermentable carbon sources include, but are notlimited to, monosaccharides such as glucose or fructose; disaccharidessuch as lactose or sucrose; oligosaccharides; polysaccharides such asstarch or cellulose; C5 sugars such as xylose and arabinose; one carbonsubstrates including methane; and mixtures thereof.

“Fermentable sugar” as used herein refers to one or more sugars capableof being metabolized by the microorganisms disclosed herein for theproduction of fermentative alcohol.

“Fermentation vessel” as used herein means the vessel in which thefermentation reaction is carried out whereby product alcohol such asbutanol is made from sugars.

“Liquefaction vessel” as used herein means the vessel in whichliquefaction is carried out. Liquefaction is the process in whicholigosaccharides are liberated from the feedstock. In some embodimentswhere the feedstock is corn, oligosaccharides are liberated from thecorn starch content during liquefaction.

“Saccharification vessel” as used herein means the vessel in whichsaccharification (i.e., the break down of oligosaccharides intomonosaccharides) is carried out. Where fermentation and saccharificationoccur simultaneously, the saccharification vessel and the fermentationvessel may be one in the same vessel.

“Sugar” as used herein refers to oligosaccharides, disaccharides,monosaccharides, and/or mixtures thereof. The term “saccharide” alsoincludes carbohydrates including starches, dextrans, glycogens,cellulose, pentosans, as well as sugars.

As used herein, “saccharification enzyme” means one or more enzymes thatare capable of hydrolyzing polysaccharides and/or oligosaccharides, forexample, alpha-1,4-glucosidic bonds of glycogen, or starch.Saccharification enzymes may include enzymes capable of hydrolyzingcellulosic or lignocellulosic materials as well.

“Undissolved solids” as used herein means non-fermentable portions offeedstock, for example, germ, fiber, and gluten.

“Product alcohol” as used herein refers to any alcohol that can beproduced by a microorganism in a fermentation process that utilizesbiomass as a source of fermentable carbon substrate. Product alcoholsinclude, but are not limited to, C₁ to C₈ alkyl alcohols. In someembodiments, the product alcohols are C₂ to C₈ alkyl alcohols. In otherembodiments, the product alcohols are C₂ to C₅ alkyl alcohols. It willbe appreciated that C₁ to C₈ alkyl alcohols include, but are not limitedto, methanol, ethanol, propanol, butanol, and pentanol. Likewise C₂ toC₈ alkyl alcohols include, but are not limited to, ethanol, propanol,butanol, and pentanol. “Alcohol” is also used herein with reference to aproduct alcohol.

“Butanol” as used herein refers with specificity to the butanol isomers1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol, and/or isobutanol(iBuOH or i-BuOH or I-BUOH, also known as 2-methyl-1-propanol), eitherindividually or as mixtures thereof. From time to time, when referringto esters of butanol, the terms “butyl esters” and “butanol esters” maybe used interchangeably.

“Propanol” as used herein refers to the propanol isomers isopropanol or1-propanol.

“Pentanol” as used herein refers to the pentanol isomers 1-pentanol,3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol,3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol.

The term “alcohol equivalent” as used herein refers to the weight ofalcohol that would be obtained by a perfect hydrolysis of an alcoholester and the subsequent recovery of the alcohol from an amount ofalcohol ester.

The term “aqueous phase titer” as used herein refers to theconcentration of a particular alcohol (e.g., butanol) in thefermentation broth.

The term “effective titer” as used herein refers to the total amount ofa particular alcohol (e.g., butanol) produced by fermentation or alcoholequivalent of the alcohol ester produced by alcohol esterification perliter of fermentation medium. For example, the effective titer ofbutanol in a unit volume of a fermentation includes: (i) the amount ofbutanol in the fermentation medium; (ii) the amount of butanol recoveredfrom the organic extractant; (iii) the amount of butanol recovered fromthe gas phase, if gas stripping is used; and (iv) the alcohol equivalentof the butanol ester in either the organic or aqueous phase.

“In Situ Product Removal (ISPR)” as used herein means the selectiveremoval of a specific fermentation product from a biological processsuch as fermentation, to control the product concentration in thebiological process as the product is produced.

“Extractant” or “ISPR extractant” as used herein means an organicsolvent used to extract any product alcohol such as butanol or used toextract any product alcohol ester produced by a catalyst from a productalcohol and a carboxylic acid or lipid. From time to time, as usedherein the term “solvent” may be used synonymously with “extractant.”For the processes described herein, extractants are water-immiscible.

The terms “water-immiscible” or “insoluble” refer to a chemicalcomponent such as an extractant or solvent, which is incapable of mixingwith an aqueous solution such as a fermentation broth, in such a manneras to form one liquid phase.

The term “aqueous phase” as used herein refers to the aqueous phase of abiphasic mixture obtained by contacting a fermentation broth with awater-immiscible organic extractant. In an embodiment of a processdescribed herein that includes fermentative extraction, the term“fermentation broth” then specifically refers to the aqueous phase inbiphasic fermentative extraction.

The term “organic phase” as used herein refers to the non-aqueous phaseof a biphasic mixture obtained by contacting a fermentation broth with awater-immiscible organic extractant.

The term “carboxylic acid” as used herein refers to any organic compoundwith the general chemical formula —COOH in which a carbon atom is bondedto an oxygen atom by a double bond to make a carbonyl group (—C═O) andto a hydroxyl group (—OH) by a single bond. A carboxylic acid may be inthe form of the protonated carboxylic acid, in the form of a salt of acarboxylic acid (e.g., an ammonium, sodium, or potassium salt), or as amixture of protonated carboxylic acid and salt of a carboxylic acid. Theterm carboxylic acid may describe a single chemical species (e.g., oleicacid) or a mixture of carboxylic acids as can be produced, for example,by the hydrolysis of biomass-derived fatty acid esters or triglycerides,diglycerides, monoglycerides, and phospholipids.

The term “fatty acid” as used herein refers to a carboxylic acid (e.g.,aliphatic monocarboxylic acid) having C₄ to C₂₈ carbon atoms (mostcommonly C₁₂ to C₂₄ carbon atoms), which is either saturated orunsaturated. Fatty acids may also be branched or unbranched. Fatty acidsmay be derived from, or contained in esterified form, in an animal orvegetable fat, oil, or wax. Fatty acids may occur naturally in the formof glycerides in fats and fatty oils or may be obtained by hydrolysis offats or by synthesis. The term fatty acid may describe a single chemicalspecies or a mixture of fatty acids. In addition, the term fatty acidalso encompasses free fatty acids.

The term “fatty alcohol” as used herein refers to an alcohol having analiphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “fatty aldehyde” as used herein refers to an aldehyde having analiphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

The term “fatty amide” as used herein refers to an amide having a long,aliphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated

The term “fatty ester” as used herein refers to an ester having a longaliphatic chain of C₄ to C₂₂ carbon atoms, which is either saturated orunsaturated.

“Native oil” as used herein refers to lipids obtained from plants (e.g.,biomass) or animals. “Plant-derived oil” as used herein refers to lipidsobtain from plants in particular. From time to time, “lipids” may beused synonymously with “oil” and “acyl glycerides.” Native oils include,but are not limited to, tallow, corn, canola, capric/caprylictriglycerides, castor, coconut, cottonseed, fish, jojoba, lard, linseed,neetsfoot, oiticica, palm, peanut, rapeseed, rice, safflower, soya,sunflower, tung, jatropha, and vegetable oil blends.

The term “separation” as used herein is synonymous with “recovery” andrefers to removing a chemical compound from an initial mixture to obtainthe compound in greater purity or at a higher concentration than thepurity or concentration of the compound in the initial mixture.

As used herein, “recombinant microorganism” refers to microorganismssuch as bacteria or yeast, that are modified by use of recombinant DNAtechniques, for example, by engineering a host cell to comprise abiosynthetic pathway such as a biosynthetic pathway to produce analcohol such as butanol.

The present invention provides extractants obtained by catalytichydrolysis of oil glycerides derived from biomass and methods ofproducing the extractants. In particular, the glycerides in biomass oilcan be catalytically hydrolyzed into fatty acids using a catalyst suchas an enzyme. The fatty acids can serve as extractants for in situremoval of a product alcohol such as butanol from a fermentation broth.Thus, the present invention also provides methods for producing aproduct alcohol such as butanol through extractive fermentation usingthe extractants that were produced from the biomass oil. The presentinvention also provides methods for catalytically hydrolyzing the oilpresent in a feedstock slurry into fatty acids prior to fermentation,whereby the oil is converted to fatty acids and the degradation of thepartition coefficient of the ISPR extractant over time that isattributable to the presence of the oil in the fermentation vessel canbe reduced. Moreover, the fatty acids obtained by hydrolysis of thefeedstock oil can serve as an ISPR extractant having a partitioncoefficient for a fermentative alcohol greater than a partitioncoefficient of the feedstock oil for the fermentative alcohol. Thefeedstock oil can be separated from the feedstock slurry prior tohydrolysis and used as an ISPR extractant, or the oil can be hydrolyzedinto fatty acids while in the feedstock slurry. Further, fatty acids asISPR extractant can be used in place of or in addition to a conventionalexogenous extractant, such as oleyl alcohol or oleic acid, therebyreducing the raw material expense associated with the exogenousextractant.

The present invention will be described with reference to the Figures.FIG. 1 illustrates an exemplary process flow diagram for production offermentative alcohol according to an embodiment of the presentinvention. As shown, a feedstock 12 can be introduced to an inlet in aliquefaction vessel 10 and liquefied to produce a feedstock slurry 16.Feedstock 12 contains hydrolysable starch that supplies a fermentablecarbon source (e.g., fermentable sugar such as glucose), and can be abiomass such as, but not limited to, rye, wheat, corn, cane, barley,cellulosic material, lignocellulosic material, or mixtures thereof, orcan otherwise be derived from a biomass. In some embodiments, feedstock12 can be one or more components of a fractionated biomass and in otherembodiments, feedstock 12 can be a milled, unfractionated biomass. Insome embodiments, feedstock 12 can be corn such as dry milled,unfractionated corn kernels, and the undissolved solids can includegerm, fiber, and gluten. The undissolved solids are non-fermentableportions of feedstock 12. For purposes of the discussion herein withreference to the embodiments shown in the Figures, feedstock 12 willoften be described as constituting milled, unfractionated corn in whichthe undissolved solids have not been separated therefrom. However, itshould be understood that the exemplary methods and systems describedherein can be modified for different feedstocks whether fractionated ornot, as apparent to one of skill in the art. In some embodiments,feedstock 12 can be high-oleic corn, such that corn oil derivedtherefrom is a high-oleic corn oil having an oleic acid content of atleast about 55 wt % oleic acid. In some embodiments, the oleic acidcontent in high-oleic corn oil can be up to about 65 wt %, as comparedwith the oleic acid content in normal corn oil which is about 24 wt %.High-oleic oil can provide some advantages for use in the methods of thepresent invention, as hydrolysis of the oil provides fatty acids havinga high oleic acid content for contacting with a fermentation broth. Insome embodiments, the fatty acids or mixtures thereof compriseunsaturated fatty acids. The presence of unsaturated fatty acidsdecreases the melting point, providing advantages for handling. Of theunsaturated fatty acids, those which are monounsaturated, that is,possessing a single carbon-carbon double bond, may provide advantageswith respect to melting point without sacrificing suitable thermal andoxidative stability for process considerations.

The process of liquefying feedstock 12 involves hydrolysis of starch infeedstock 12 into sugars including, for example, dextrins andoligosaccharides, and is a conventional process. Any known liquefyingprocesses, as well as the corresponding liquefaction vessel, normallyutilized by the industry can be used including, but not limited to, theacid process, the acid-enzyme process, or the enzyme process. Suchprocesses can be used alone or in combination. In some embodiments, theenzyme process can be utilized and an appropriate enzyme 14, forexample, alpha-amylase, is introduced to an inlet in liquefaction vessel10. Water can also be introduced to liquefaction vessel 10. In someembodiments, a saccharification enzyme, for example, glucoamylase, mayalso be introduced to liquefaction vessel 10. In additional embodiments,a lipase may also be introduced to liquefaction vessel 10 to catalyzethe conversion of one or more components of the oil to fatty acids.

Feedstock slurry 16 produced from liquefying feedstock 12 includessugar, oil 26, and undissolved solids derived from the biomass fromwhich feedstock 12 was formed. In some embodiments, the oil is in anamount of about 0 wt % to at least about 2 wt % of the fermentationbroth composition. In some embodiments, the oil is in an amount of atleast about 0.5 wt % of the feedstock. Feedstock slurry 16 can bedischarged from an outlet of liquefaction vessel 10. In someembodiments, feedstock 12 is corn or corn kernels and therefore,feedstock slurry 16 is a corn mash slurry.

A catalyst 42 can be added to feedstock slurry 16. Catalyst 42 iscapable of hydrolyzing glycerides in oil 26 to free fatty acids (FFA)28. For example, when feedstock 12 is corn, then oil 26 is thefeedstock's constituent corn oil, and the free fatty acids 28 are cornoil fatty acids (COFA). Thus, after introduction of catalyst 42 tofeedstock slurry 16, at least a portion of the glycerides in oil 26 arehydrolyzed to FFA 28, resulting in a feedstock slurry 18 having FFA 28and catalyst 42. The resulting acid/oil composition from hydrolyzing oil26 is typically at least about 17 wt % FFA. In some embodiments, theresulting acid/oil composition from hydrolyzing oil 26 is at least about20 wt % FFA, at least about 25 wt % FFA, at least about 30 wt % FFA, atleast about 35 wt % FFA, at least about 40 wt % FFA, at least about 45wt % FFA, at least about 50 wt % FFA, at least about 55 wt % FFA, atleast about 60 wt % FFA, at least about 65 wt % FFA, at least about 70wt % FFA, at least about 75 wt % FFA, at least about 80 wt % FFA, atleast about 85 wt % FFA, at least about 90 wt % FFA, at least about 95wt % FFA, or at least about 99 wt % FFA. In some embodiments, theconcentration of the fatty acid (such as carboxylic acid) in thefermentation vessel exceeds the solubility limit in the aqueous phaseand results in the production a two-phase fermentation mixturecomprising an organic phase and an aqueous phase. In some embodiments,the concentration of carboxylic acid (or fatty acid) in the fermentationbroth is typically not greater than about 0.8 g/L and is limited by thesolubility of the carboxylic acid (or fatty acid) in the broth.

In some embodiments, catalyst 42 can be one or more enzymes, forexample, hydrolase enzymes such as lipase enzymes. Lipase enzymes usedmay be derived from any source including, for example, Absidia,Achromobacter, Aeromonas, Alcaligenes, Alternaria, Aspergillus,Achromobacter, Aureobasidium, Bacillus, Beauveria, Brochothrix, Candida,Chromobacter, Coprinus, Fusarium, Geotricum, Hansenula, Humicola,Hyphozyma, Lactobacillus, Metarhizium, Mucor, Nectria, Neurospora,Paecilomyces, Penicillium, Pseudomonas, Rhizoctonia, Rhizomucor,Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Sus,Sporobolomyces, Thermomyces, Thiarosporella, Trichoderma, Verticillium,and/or a strain of Yarrowia. In a preferred aspect, the source of thelipase is selected from the group consisting of Absidia blakesleena,Absidia corymbifera, Achromobacter iophagus, Alcaligenes sp., Alternariabrassiciola, Aspergillus flavus, Aspergillus niger, Aspergillustubingensis, Aureobasidium pullulans, Bacillus pumilus, Bacillusstrearothermophilus, Bacillus subtilis, Brochothrix thermosohata,Candida cylindracea (Candida rugosa), Candida paralipolytica, CandidaAntarctica lipase A, Candida antartica lipase B, Candida ernobii,Candida deformans, Chromobacter viscosum, Coprinus cinerius, Fusariumoxysporum, Fusarium solani, Fusarium solani pisi, Fusarium roseumculmorum, Geotricum penicillatum, Hansenula anomala, Humicolabrevispora, Humicola brevis var. thermoidea, Humicola insolens,Lactobacillus curvatus, Rhizopus oryzae, Penicillium cyclopium,Penicillium crustosum, Penicillium expansum, Penicillium sp. I,Penicillium sp. II, Pseudomonas aeruginosa, Pseudomonas alcaligenes,Pseudomonas cepacia (syn. Burkholderia cepacia), Pseudomonasfluorescens, Pseudomonas fragi, Pseudomonas maltophilia, Pseudomonasmendocina, Pseudomonas mephitica lipolytica, Pseudomonas alcaligenes,Pseudomonas plantari, Pseudomonas pseudoalcaligenes, Pseudomonas putida,Pseudomonas stutzeri, and Pseudomonas wisconsinensis, Rhizoctoniasolani, Rhizomucor miehei, Rhizopus japonicus, Rhizopus microsporus,Rhizopus nodosus, Rhodosporidium toruloides, Rhodotorula glutinis,Saccharomyces cerevisiae, Sporobolomyces shibatanus, Sus scrofa,Thermomyces lanuginosus (formerly Humicola lanuginose), Thiarosporellaphaseolina, Trichoderma harzianum, Trichoderma reesei, and Yarrowialipolytica. In a further preferred aspect, the lipase is selected fromthe group consisting of Thermomcyces lanuginosus lipase, Aspergillus sp.lipase, Aspergillus niger lipase, Aspergillus tubingensis lipase,Candida antartica lipase B, Pseudomonas sp. lipase, Penicilliumroqueforti lipase, Penicillium camembertii lipase, Mucor javanicuslipase, Burkholderia cepacia lipase, Alcaligenes sp. lipase, Candidarugosa lipase, Candida parapsilosis lipase, Candida deformans lipase,lipases A and B from Geotrichum candidum, Neurospora crassa lipase,Nectria haematococca lipase, Fusarium heterosporum lipase Rhizopusdelemar lipase, Rhizomucor miehei lipase, Rhizopus arrhizus lipase, andRhizopus oryzae lipase. Suitable commercial lipase preparations suitableas enzyme catalyst 42 include, but are not limited to Lipolase® 100 L,Lipex® 100L, Lipoclean® 2000T, Lipozyme® CALB L, Novozyme® CALA L, andPalatase 20000L, available from Novozymes, or from Pseudomonasfluorescens, Pseudomonas cepacia, Mucor miehei, hog pancreas, Candidacylindracea, Rhizopus niveus, Candida antarctica, Rhizopus arrhizus orAspergillus available from SigmaAldrich.

Phospholipases are enzymes that hydrolyze the ester bonds ofphospholipids, but many phospholipases also can hydrolyze triglycerides,diglycerides, and monoglycerides (lipid acyl hydrolase (LAH) activity).As used herein, the term “phospholipase” encompasses enzymes having anyphospholipase activity, for example, cleaving a glycerolphosphate esterlinkage (catalyzing hydrolysis of a glycerolphosphate ester linkage),for example, in an oil, such as a crude oil or a vegetable oil. Thephospholipase activity of the invention can generate a water extractablephosphorylated base and a diglyceride. The phospholipase activity cancomprise a phospholipase C (PLC) activity; a PI-PLC activity, aphospholipase A (PLA) activity such as a phospholipase A1 orphospholipase A2 activity; a phospholipase B (PLB) activity such as aphospholipase B1 or phospholipase B2 activity, includinglysophospholipase (LPL) activity and/or lysophospholipase-transacylase(LPT A) activity; a phospholipase D (PLD) activity such as aphospholipase DI or a phospholipase D2 activity; and/or a patatinactivity or any combination thereof. The term “phospholipase” alsoencompasses enzymes having lysophospholipase activity, where the twosubstrates of this enzyme are 2-lysophosphatidylcholine and H₂O, andwhere its two products are glycerophosphocholine and carboxylate.Phospholipase AI (PLA1) enzymes remove the 1-position fatty acid toproduce free fatty acid and 1-lyso-2-acylphospholipid. Phospholipase A2(PLA2) enzymes remove the 2-position fatty acid to produce free fattyacid and l-acyl-2-lysophospholipid. PLA1 and PLA2 enzymes can be intra-or extra-cellular, membrane-bound or soluble. Phospholipase C (PLC)enzymes remove the phosphate moiety to produce 1,2 diacylglycerol and aphosphate ester. Phospholipase D (PLD) enzymes produce1,2-diacylglycerophosphate and base group. A phospholipase useful in thepresent invention may be obtained from a variety of biological sources,for example, but not limited to, filamentous fungal species within thegenus Fusarium, such as a strain of F. culmorum, F. heterosporum, F.solani, or F. oxysporum; or a filamentous fungal species within thegenus Aspergillus, such as a strain of Aspergillus awamori, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus niger or Aspergillusoryzae. Also useful in the present invention are Thermomyces lanuginosusphospholipase variants such as the commercial product Lecitase® Ultra(Novozymes A'S, Denmark). One or more phospholipases may be applied aslyophilized powder, immobilized or in aqueous solution.

After at least a portion of the glycerides are hydrolyzed, in someembodiments, catalyst 42 can be inactivated. Any method known in the artcan be used to render catalyst 42 inactive. For example, in someembodiments, catalyst 42 can be inactivated by the application of heat,by adjusting the pH of the reaction mass to a pH where catalyst 42 isirreversibly inactivated, and/or by adding a chemical or biochemicalspecies capable of selectively inactivating the catalyst activity. Asshown, for example, in the embodiment of FIG. 1, heat q is applied tofeedstock slurry 18, whereby catalyst 42 becomes inactive. Theapplication of heat q can be applied to feedstock slurry 18 beforefeedstock slurry 18 is fed to a fermentation vessel 30. Heat-treatedfeedstock slurry 18 (with inactive catalyst 42) is then introduced intoa fermentation vessel 30 along with a microorganism 32 to be included ina fermentation broth held in fermentation vessel 30. Alternatively,feedstock slurry 18 can be fed to fermentation vessel 30 and subjectedto heat q while in the fermentation vessel, before fermentation vesselinoculation of microorganism 32. For example, in some embodiments,catalyst inactivation treatment can be achieved by heating feedstockslurry 18 with heat q to temperature of at least about 75° C. for atleast about 5 minutes, at least about 75° C. for at least about 10minutes, at least about 75° C. for at least about 15 minutes, at leastabout 80° C. for at least about 5 minutes, at least about 80° C. for atleast about 10 minutes, at least about 80° C. for at least about 15minutes, at least about 85° C. for at least about 5 minutes, at leastabout 85° C. for at least about 10 minutes, or at least about 85° C. forat least about 15 minutes. In some embodiments, after being subject toheat q, feedstock slurry 18 is cooled to an appropriate temperature forfermentation prior to introduction to fermentation vessel 30 (or priorto fermentation vessel inoculation in the case that the application ofheat q is conducted in the fermentation vessel). For example, in someembodiments, the temperature of feedstock slurry 18 is about 30° C.prior to contacting with a fermentation broth.

Inactivation of catalyst 42 is preferred when it is desirable to preventcatalyst 42 from esterifying alcohol with fatty acids 28 in thefermentation vessel. In some embodiments, production of an alcohol esterby esterification of product alcohol in a fermentation medium with anorganic acid (e.g., fatty acid) and a catalyst (e.g., lipase) isdesirable, as further described in co-pending, commonly owned U.S.Provisional Application Ser. No. 61/368,429, filed on Jul. 28, 2010;U.S. Provisional Application Ser. No. 61/379,546, filed on Sep. 2, 2010;and U.S. Provisional Application Ser. No. 61/440,034, filed on Feb. 7,2011, all incorporated herein in its entirety by reference thereto. Forexample, for butanol production, active catalyst 42 in fermentationvessel (introduced via slurry 18) can catalyze the esterification of thebutanol with fatty acids 28 (introduced via slurry 18) to form fattyacid butyl esters (FABE) in situ.

Fermentation vessel 30 is configured to ferment slurry 18 to produce aproduct alcohol such as butanol. In particular, microorganism 32metabolizes the fermentable sugar in slurry 18 and excretes a productalcohol. Microorganism 32 is selected from the group of bacteria,cyanobacteria, filamentous fungi, and yeasts. In some embodiments,microorganism 32 can be a bacteria such as E. coli. In some embodiments,microorganism 32 can be a fermentative recombinant microorganism. Theslurry can include sugar, for example, in the form of oligosaccharides,and water, and can comprise less than about 20 g/L of monomeric glucose,more preferably less than about 10 g/L or less than about 5 g/L ofmonomeric glucose. Suitable methodology to determine the amount ofmonomeric glucose is well known in the art. Such suitable methods knownin the art include HPLC.

In some embodiments, slurry 18 is subjected to a saccharificationprocess in order to break the complex sugars (e.g., oligosaccharides) inslurry 18 into monosaccharides that can be readily metabolized bymicroorganism 32. Any known saccharification process that is routinelyutilized by the industry can be used including, but not limited to, theacid process, the acid-enzyme process, or the enzyme process. In someembodiments, simultaneous saccharification and fermentation (SSF) canoccur inside fermentation vessel 30, as shown in FIG. 1. In someembodiments, an enzyme 38, such as glucoamylase, can be introduced to aninlet in fermentation vessel 30 in order to breakdown the starch oroligosaccharides to glucose capable of being metabolized bymicroorganism 32.

Optionally, ethanol 33 may be supplied to fermentation vessel 30 to beincluded in the fermentation broth. In some embodiments, when arecombinant microorganism having a butanol biosynthetic pathway is usedas microorganism 32 for butanol production, microorganism 32 may requiresupplementation of a 2-carbon substrate (e.g., ethanol) to survive andgrow. Thus, in some embodiments, ethanol 33 may be supplied tofermentation vessel 30.

However, it has been surprisingly found that methods of the presentinvention, in which free fatty acid (e.g., FFA 28) is present in thefermentation vessel, can allow reduction of the amount of ethanol 33typically supplied for a given recombinant microorganism withoutdetriment to the vitality of the recombinant microorganism. Further, insome embodiments, the methods of the present invention provide that thealcohol (e.g., butanol) production rate without ethanol supplementationto be comparable with the production rate that can be realized whenethanol 33 is supplemented. As further demonstrated by the comparativeexamples presented in Examples 1-14 below, the butanol production ratewhen fatty acid but not ethanol is in the fermentation vessel can begreater than the butanol production rate when neither fatty acid norethanol is in the fermentation vessel. Thus, in some embodiments, theamount of ethanol 33 supplementation is reduced compared to conventionalprocesses. For example, a typical amount of ethanol added to afermentation vessel for microorganisms requiring supplementation of a2-carbon substrate is about 5 g/L anhydrous ethanol (i.e., 5 g anhydrousethanol per liter of fermentation medium). In some embodiments, thebutanol fermentation is not supplemented with any ethanol 33. In thelatter case, the stream of ethanol 33 is entirely omitted from thefermentation vessel. Thus, in some embodiments of the present invention,it is possible to reduce or eliminate the cost associated withsupplemental ethanol 33, as well as the inconvenience associated withstoring vats of ethanol 33 and supplying it to the fermentation vesselduring butanol fermentation.

Moreover, regardless of ethanol supplementation, in some embodiments,the methods of the present invention can provide a higher rate ofglucose uptake by microorganism 32 by virtue of the presence of fattyacids during the fermentation. The fatty acids can be introduced intofermentation vessel 30 as carboxylic acid 28, hydrolyzed from suppliedoil 26, and/or derived from hydrolysis of constituent biomass oil ofslurry 16. Methods for producing a product alcohol from a fermentationprocess in which fatty acids are produced at a step in the process andare contacted with microorganism cultures in a fermentation vessel forimproving microorganism growth rate and glucose consumption aredescribed in co-pending, commonly owned U.S. Provisional ApplicationSer. No. 61/368,451, filed on Jul. 28, 2010, which is incorporatedherein in its entirety by reference thereto.

In fermentation vessel 30, alcohol is produced by microorganism 32. Insitu product removal (ISPR) can be utilized to remove the productalcohol from the fermentation broth. In some embodiments, ISPR includesliquid-liquid extraction. Liquid-liquid extraction can be performedaccording to the processes described in U.S. Patent ApplicationPublication No. 2009/0305370, the disclosure of which is herebyincorporated in its entirety. U.S. Patent Application Publication No.2009/0305370 describes methods for producing and recovering butanol froma fermentation broth using liquid-liquid extraction, the methodscomprising the step of contacting the fermentation broth with awater-immiscible extractant to form a two-phase mixture comprising anaqueous phase and an organic phase. Typically, the extractant can be anorganic extractant selected from the group consisting of saturated,mono-unsaturated, poly-unsaturated (and mixtures thereof) C₁₂ to C₂₂fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fattyacids, C₁₂ to C₂₂ fatty aldehydes, C₁₂ to C₂₂ fatty amides,triglycerides, and mixtures thereof, which contacts a fermentation brothand to form a two-phase mixture comprising an aqueous phase and anorganic phase. The extractant may also be an organic extractant selectedfrom the group consisting of saturated, mono-unsaturated,poly-unsaturated (and mixtures thereof) C₄ to C₂₂ fatty alcohols, C₄ toC₂₈ fatty acids, esters of C₄ to C₂₈ fatty acids, C₄ to C₂₂ fattyaldehydes, C₄ to C₂₂ fatty amides, and mixtures thereof, which contactsa fermentation broth and to form a two-phase mixture comprising anaqueous phase and an organic phase. Free fatty acids 28 from slurry 18can also serve as an ISPR extractant 28. For example, when free fattyacids 28 are corn oil fatty acids (COFA), ISPR extractant 28 is COFA.ISPR extractant (FFA) 28 contacts the fermentation broth and forms atwo-phase mixture comprising an aqueous phase 34 and an organic phase.The product alcohol present in the fermentation broth preferentiallypartitions into the organic phase to form an alcohol-containing organicphase 36. In some embodiments, fermentation vessel 30 has one or moreinlets for receiving one or more additional ISPR extractants 29 whichform a two-phase mixture comprising an aqueous phase and an organicphase, with the product alcohol partitioning into the organic phase.

The biphasic mixture can be removed from fermentation vessel 30 asstream 39 and introduced into a vessel 35, in which thealcohol-containing organic phase 36 is separated from the aqueous phase34. The alcohol-containing organic phase 36 is separated from theaqueous phase 34 of the biphasic mixture stream 39 using methods knownin the art including, but not limited to, siphoning, aspiration,decantation, centrifugation, using a gravity settler, membrane-assistedphase splitting, and the like. All or part of the aqueous phase 34 canbe recycled into fermentation vessel 30 as fermentation medium (asshown), or otherwise discarded and replaced with fresh medium, ortreated for the removal of any remaining product alcohol and thenrecycled to fermentation vessel 30. Then, the alcohol-containing organicphase 36 is treated in a separator 50 to recover product alcohol 54, andthe resulting alcohol-lean extractant 27 can then be recycled back intofermentation vessel 30, usually in combination with fresh FFA 28 fromslurry 18 and/or with fresh extractant 29 for further extraction of theproduct alcohol. Alternatively, fresh FFA 28 (from slurry 18) and/orextractant 29 can be continuously added to the fermentation vessel toreplace the ISPR extractant(s) removed in biphasic mixture stream 39.

In some embodiments, any additional ISPR extractant 29 can be anexogenous organic extractant such as oleyl alcohol, behenyl alcohol,cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol,1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid,methyl myristate, methyl oleate, undecanal, lauric aldehyde,20-methylundecanal, and mixtures thereof. In some embodiments, ISPRextractant 29 can be a carboxylic acid and in some embodiments, ISPRextractant 29 can be a fatty acid. In some embodiments, the carboxylicacid or fatty acid can have 4 to 28 carbons, 4 to 22 carbons in otherembodiments, 8 to 22 carbons in other embodiments, 10 to 28 carbons inother embodiments, 7 to 22 carbons in other embodiments, 12 to 22carbons in other embodiments, 4 to 18 carbons in other embodiments, 12to 22 carbons in other embodiments, and 12 to 18 carbons in still otherembodiments. In some embodiments, ISPR extractant 29 is one or more ofthe following fatty acids: azaleic, capric, caprylic, castor, coconut(i.e., as a naturally-occurring combination of fatty acids, includinglauric, myrisitic, palmitic, caprylic, capric, stearic, caproic,arachidic, oleic, and linoleic, for example), dimer, isostearic, lauric,linseed, myristic, oleic, olive, palm oil, palmitic, palm kernel,peanut, pelargonic, ricinoleic, sebacic, soya, stearic acid, tall oil,tallow, #12 hydroxy stearic, or any seed oil. In some embodiments, ISPRextractant 29 is one or more of diacids, azelaic, dimer and sebacicacid. Thus, in some embodiments, ISPR extractant 29 can be a mixture oftwo or more different fatty acids. In some embodiments, ISPR extractant29 can be a fatty acid derived from chemical or enzymatic hydrolysis ofglycerides derived from native oil. For example, in some embodiments,ISPR extractant 29 can be free fatty acids 28′ obtained by enzymatichydrolysis of native oil such as biomass lipids as later described withreference to the embodiment of FIG. 5. In some embodiments, ISPRextractant 29 can be a fatty acid extractant selected from the groupconsisting of fatty acids, fatty alcohols, fatty amides, fatty acidmethyl esters, lower alcohol esters of fatty acids, fatty acid glycolesters, hydroxylated triglycerides, and mixtures thereof, obtained fromchemical conversion of native oil such as biomass lipids as describedfor example in co-pending, commonly owned U.S. Provisional ApplicationSer. No. 61/368,436, filed on Jul. 28, 2010. In such embodiments, thebiomass lipids for producing extractant 29 can be from a same ordifferent biomass source from which feedstock 12 is obtained. Forexample, in some embodiments, the biomass lipids for producingextractant 29 can be derived from soya, whereas the biomass source offeedstock 12 is corn. Any possible combination of different biomasssources for extractant 29 versus feedstock 12 can be used, as should beapparent to one of skill in the art. In some embodiments, additionalISPR extractant 29 includes COFA.

In situ extractive fermentation can be carried out in a batch mode or acontinuous mode in fermentation vessel 30. For in situ extractivefermentation, the organic extractant can contact the fermentation mediumat the start of the fermentation forming a biphasic fermentation medium.Alternatively, the organic extractant can contact the fermentationmedium after the microorganism has achieved a desired amount of growth,which can be determined by measuring the optical density of the culture.Further, the organic extractant can contact the fermentation medium at atime at which the product alcohol level in the fermentation mediumreaches a preselected level. In the case of butanol production, forexample, the ISPR extractant can contact the fermentation medium at atime before the butanol concentration reaches a level which would betoxic to the microorganism. After contacting the fermentation mediumwith the ISPR extractant, the butanol product partitions into theextractant, decreasing the concentration in the aqueous phase containingthe microorganism, thereby limiting the exposure of the productionmicroorganism to the inhibitory butanol product.

The volume of the ISPR extractant to be used depends on a number offactors including the volume of the fermentation medium, the size of thefermentation vessel, the partition coefficient of the extractant for thebutanol product, and the fermentation mode chosen, as described below.The volume of the extractant can be about 3% to about 60% of thefermentation vessel working volume. Depending on the efficiency of theextraction, the aqueous phase titer of butanol in the fermentationmedium can be, for example, from about 1 g/L to about 85 g/L, from about10 g/L to about 40 g/L, from about 10 g/L to about 20 g/L, from about 15g/L to about 50 g/L or from about 20 g/L to about 60 g/L. In someembodiments, the resulting fermentation broth after alcoholesterification can comprise free (i.e., unesterified) alcohol and insome embodiments, the concentration of free alcohol in the fermentationbroth after alcohol esterification is not greater than 1, 3, 6, 10, 15,20, 25, 30 25, 40, 45, 50, 55, or 60 g/L when the product alcohol isbutanol, or when the product alcohol is ethanol, the concentration offree alcohol in the fermentation broth after alcohol esterification isnot greater than 15, 20, 25, 30 25, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 g/L. Without being held to theory, it is believedthat higher butanol titer may obtained with the extractive fermentationmethod, in part, from the removal of the toxic butanol product from thefermentation medium, thereby keeping the level below that which is toxicto the microorganism.

In a batchwise mode of in situ extractive fermentation, a volume oforganic extractant is added to the fermentation vessel and theextractant is not removed during the process. This mode requires alarger volume of organic extractant to minimize the concentration of theinhibitory butanol product in the fermentation medium. Consequently, thevolume of the fermentation medium is less and the amount of productproduced is less than that obtained using the continuous mode. Forexample, the volume of the extractant in the batchwise mode can be 20%to about 60% of the fermentation vessel working volume in oneembodiment, and about 30% to about 60% in another embodiment.

Gas stripping (not shown) can be used concurrently with the ISPRextractant to remove the product alcohol from the fermentation medium.

In the embodiment of FIG. 1, the product alcohol is extracted from thefermentation broth in situ, with the separation of the biphasic mixture39 occurring in a separate vessel 35. In some embodiments, separation ofthe biphasic mixture 39 can occur in the fermentation vessel, as shownin the embodiments of later described FIGS. 2 and 3 in which thealcohol-containing organic phase stream 36 exits directly fromfermentation vessel 30. Aqueous phase stream 34 can also exit directlyfrom fermentation vessel 30, be treated for the removal of any remainingproduct alcohol and recycled, or discarded and replaced with freshfermentation medium. The extraction of the product alcohol by theorganic extractant(s) can be done with or without the removal of themicroorganism from the fermentation broth. The microorganism can beremoved from the fermentation broth by means known in the art including,but not limited to, filtration or centrifugation. For example, aqueousphase stream 34 can include microorganism 32 such as yeast.Microorganism 32 can be easily separated from the aqueous phase stream,for example, in a centrifuge (not shown). Microorganism 32 can then berecycled to fermentation vessel 30 which over time can increase theproduction rate of alcohol production, thereby resulting in an increasein the efficiency of the alcohol production.

In a continuous mode of in situ extractive fermentation, the volume ofthe extractant can be about 3% to about 50% of the fermentation vesselworking volume in one embodiment, about 3% to about 30% in anotherembodiment, 3% to about 20% in another embodiment; and 3% to about 10%in another embodiment. Because the product is continually removed fromthe reactor, a smaller volume of extractant is required enabling alarger volume of the fermentation medium to be used.

As an alternative to in situ extractive fermentation, the productalcohol can be extracted from the fermentation broth downstream offermentation vessel 30. In such an instance, the fermentation broth canbe removed from fermentation vessel 30 and introduced into vessel 35.Extractant 28 can then be introduced into vessel 35 and contacted withthe fermentation broth to obtain biphasic mixture 39 in vessel 35, whichis then separated into the organic and aqueous phases 36 and 34.Alternatively, extractant 28 can be added to the fermentation broth in aseparate vessel (not shown) prior to introduction to vessel 35.

As a non-limiting prophetic example, with reference to the embodiment ofFIG. 1, an aqueous suspension of ground whole corn (as feedstock 12),which can nominally contain about 4 wt % corn oil, can be treated withamylase (as liquefaction enzyme 14) at about 85° C. to 120° C. for 30minutes to 2 hours, and the resulting liquefied mash 16 cooled tobetween 65° C. and 30° C. and treated with 0.1 ppm to 10 ppm (in someembodiments, 0.5 ppm to 1.0 ppm) of lipase (as catalyst 42) at pH 4.5 to7.5 (in some embodiments, between pH 5.5 and 6.5) for sufficient time toproduce from at least 30% to as high as at least 99% conversion of theavailable fatty acid content in lipids to free fatty acids. Optionally,the liquefied and lipase-treated mash 18 can be heated to inactivatelipase 42 prior to fermentation. Mash 18 can be cooled to about 30° C.(e.g., using a heat-exchanger) and loaded to fermentation vessel 30 atabout 25% to 30 wt % dry corn solids. Saccharification of the liquefiedmash 18 during fermentation by the addition of glucoamylase (assaccharification enzyme 38) can result in the production of glucose. Theresulting fermentation broth can contain significantly less than theamount of corn oil (e.g., about 1.2 wt % corn oil) that can be presentin a fermentation broth using a liquefied mash that has not been treatedwith lipase 42. In particular, the lipase 42 treatment can result in theconversion of corn oil lipids 26 (triglycerides (TG)) into COFA as FFA28 (and some diglycerides (DG) or monoglycerides (MG)), decreasing therate of build-up of lipids 26 in any ISPR extractant 29 (e.g., oleylalcohol), and dissolution of COFA 28 into organic phase 36 during ISPRshould not decrease the partition coefficient of butanol in organicphase 36 as much as would the dissolution of lipids (TG) into theorganic phase 36.

In some embodiments, the system and processes of FIG. 1 can be modifiedsuch that feedstock slurry 16 (having oil 26) and catalyst 42 areintroduced and contacted in fermentation vessel 30 so as to produceslurry 18 (having FFA 28). The fermentation vessel temperature can thenbe raised to heat inactivate catalyst 42. The fermentation vesseltemperature can then be reduced, and the fermentation vessel can beinoculated with microorganism 32, whereby the sugars of slurry 18 can befermented to produce a product alcohol.

In some embodiments, the system and processes of FIG. 1 can be modifiedsuch that simultaneous saccharification and fermentation (SSF) infermentation vessel 30 is replaced with a separate saccharificationvessel 60 (see FIG. 2) prior to fermentation vessel 30, as should beapparent to one of skill in the art. Thus, slurry 18 can be saccharifiedeither before fermentation or during fermentation in an SSF process. Itshould also be apparent that catalyst 42 for hydrolysis of feedstock oil26 can be introduced before, after, or contemporaneously withsaccharification enzyme 38. Thus, in some embodiments, addition ofenzyme 38 and catalyst 42 can be stepwise (e.g., catalyst 42, thenenzyme 38, or vice versa), or substantially simultaneous (i.e., atexactly the same time as in the time it takes for a person or a machineto perform the addition in one stroke, or one enzyme/catalystimmediately following the other catalyst/enzyme as in the time it takesfor a person or a machine to perform the addition in two strokes).

For example, as shown in the embodiment of FIG. 2, the system andprocesses of FIG. 1 can be modified such that simultaneoussaccharification and fermentation (SSF) in fermentation vessel 30 isreplaced with a separate saccharification vessel 60 prior tofermentation vessel 30. FIG. 2 is substantially identical to FIG. 1except for the inclusion of a separate saccharification vessel 60receiving enzyme 38, with catalyst 42 being introduced to a liquefied,saccharified feedstock stream 62. Feedstock slurry 16 is introduced intosaccharification vessel 60 along with enzyme 38 such as glucoamylase,whereby sugars in the form of oligosaccharides in slurry 16 can bebroken down into monosaccharides. A liquefied, saccharified feedstockstream 62 exits saccharification vessel 60 to which catalyst 42 isintroduced. Feedstock stream 62 includes monosaccharides, oil 26, andundissolved solids derived from the feedstock. Oil 26 is hydrolyzed bythe introduction of catalyst 42 resulting in a liquefied, saccharifiedfeedstock slurry 64 having free fatty acids 28 and catalyst 42.

Alternatively, in some embodiments, catalyst 42 can be added withsaccharification enzyme 38 to simultaneously produce glucose andhydrolyze oil lipids 26 to free fatty acids 28. The addition of enzyme38 and catalyst 42 can be stepwise (e.g., catalyst 42, then enzyme 38,or vice versa) or simultaneous. Alternatively, in some embodiments,slurry 62 can be introduced to fermentation vessel with catalyst 42being added directly to the fermentation vessel 30.

In the embodiment of FIG. 2, heat q is applied to feedstock slurry 64,whereby catalyst 42 becomes inactive, and heat-treated slurry 64 is thenintroduced to fermentation vessel 30 along with alcohol-producingmicroorganism 32, which metabolizes the monosaccharides to produce aproduct alcohol (e.g., butanol). Alternatively, slurry 64 can be fed tofermentation vessel 30 and subjected to heat q while in the fermentationvessel, before inoculation of microorganism 32.

As described above with reference to FIG. 1, free fatty acids 28 canalso serve as an ISPR extractant for preferentially partitioning theproduct alcohol from the aqueous phase. In some embodiments, one or moreadditional ISPR extractants 29 can also be introduced into fermentationvessel 30. Separation of the biphasic mixture occurs in fermentationvessel 30, whereby alcohol-containing organic phase stream 36 andaqueous phase stream 34 exit directly from fermentation vessel 30.Alternatively, separation of the biphasic mixture can be conducted in aseparate vessel 35 as provided in the embodiments of FIG. 1. Theremaining process operations of the embodiment of FIG. 2 are identicalto FIG. 1 and therefore, will not be described in detail again.

In still other embodiments of the present invention, oil 26 derived fromfeedstock 12 can be catalytically hydrolyzed into FFA 28 either prior toor during liquefaction. For example, in the embodiment of FIG. 3,feedstock 12 having oil 26 is fed to liquefaction vessel 10, along withcatalyst 42 for hydrolysis of at least a portion of the glycerides inoil 26 into FFA 28. Enzyme 14 (e.g., alpha-amylase) for hydrolyzing thestarch in feedstock 12 can also be introduced to vessel 10 to produce aliquefied feedstock. The addition of enzyme 14 and catalyst 42 can bestepwise or simultaneous. For example, catalyst 42 can be introduced,and then enzyme 14 can be introduced after at least a portion of oil 26has been hydrolyzed. Alternatively, enzyme 14 can be introduced, andthen catalyst 42 can be introduced. The liquefaction process can involvethe application of heat q. In such embodiments, it is preferred thatcatalyst 42 is introduced prior to or during liquefaction when theprocess temperature is below that which inactivates catalyst 42, so thatoil 26 can be hydrolyzed. Thereafter, application of heat q can providea two-fold purpose of liquefaction and inactivation of catalyst 42.

In any case, oil 26 in feedstock 12 is converted to FFA 28 inliquefaction vessel 10, such that biphasic feedstock slurry 18 exitsliquefaction vessel 10. Biphasic slurry 18 includes both an organicphase of FFA 28 as well as sugar, water, and undissolved solids of anaqueous phase. In some embodiments, the aqueous phase can includeglycerol (glycerin) from converting the glycerides in the oil to fattyacids. In some embodiments, such glycerol, if present, can be removedfrom the stream 18 prior to introduction into fermentation vessel 30.

With reference to FIG. 3, biphasic stream 18 is contacted with thefermentation broth in fermentation vessel 30 to form a biphasic mixture.In fermentation vessel 30, product alcohol produced by SSF partitionsinto the organic phase including FFA 28. Alternatively, in someembodiments, the process can be modified to include a separatesaccharification vessel as discussed in connection with FIG. 2.Separation of the biphasic mixture occurs in fermentation vessel 30,whereby alcohol-containing organic phase stream 36 and aqueous phasestream 34 exit directly from fermentation vessel 30. Alternatively,separation of the biphasic mixture can be conducted in a separate vessel35 as provided in the embodiments of FIG. 1. Optionally, one or moreadditional extractants 29 can be introduced into fermentation vessel 30to form an organic phase that preferentially partitions the productalcohol from the aqueous phase. Alcohol-containing organic phase 36 canbe introduced to separator 50 for recovery of product alcohol 54 andoptional recycle of recovered extractant 27 as shown in FIG. 1. Theremaining process operations of the embodiment of FIG. 3 can beidentical to the previously described figures and therefore, will not bedescribed in detail again.

In some embodiments, including any of the earlier described embodimentswith respect to FIGS. 1-3, undissolved solids can be removed from thefeedstock slurry prior to introduction into fermentation vessel 30. Forexample, as shown in the embodiment of FIG. 4, feedstock slurry 16 isintroduced into an inlet of a separator 20 which is configured todischarge the undissolved solids as a solid phase or wet cake 24. Forexample, in some embodiments, separator 20 may include a filter press,vacuum filtration, or a centrifuge for separating the undissolved solidsfrom feedstock slurry 16. Optionally, in some embodiments, separator 20can also be configured to remove some, or substantially all, of oil 26present in feedstock slurry 16. In such embodiments, separator 20 can beany suitable separator known in the art for removing oil from an aqueousfeedstream including, but not limited to, siphoning, decantation,aspiration, centrifugation, using a gravity settler, membrane-assistedphase splitting, and the like. The remaining feedstock including thesugar and water is discharged as an aqueous stream 22 to fermentationvessel 30.

In some embodiments, separator 20 removes oil 26 but not undissolvedsolids. Thus, aqueous stream 22 fed to fermentation vessel 30 includesundissolved solids. For example, in some embodiments, separator 20includes a tricanter centrifuge 20 that agitates or spins feedstockslurry 16 to produces a centrifuge product comprising an aqueous layercontaining the sugar and water (i.e., stream 22), a solids layercontaining the undissolved solids (i.e., wet cake 24), and an oil layer(i.e., oil stream 26). In such a case, catalyst 42 can be contacted withthe removed oil 26 to produce a stream of FFA 28 including catalyst 42,as shown in FIG. 4. Heat q can then be applied to the stream of FFA 28,whereby catalyst 42 becomes inactive. The stream of FFA 28 and inactivecatalyst 42 can then be introduced into fermentation vessel 30, alongwith stream 22 and microorganism 32. Alternatively, FFA 28 and activecatalyst 42 can be fed to fermentation vessel 30 from vessel 40, andactive catalyst 42 can thereafter be subjected to heat q and inactivatedwhile in the fermentation vessel, before inoculation of microorganism32.

FFA 28 can serve as ISPR extractant 28 and forms a biphasic mixture infermentation vessel 30. Product alcohol produced by SSF partitions intoorganic phase 36 constituted by FFA 28. In some embodiments, one or moreadditional ISPR extractants 29 can also be introduced into fermentationvessel 30. Thus, oil 26 (e.g., from feedstock) can be catalyticallyhydrolyzed to FFA 28, thereby decreasing the rate of build-up of lipidsin an ISPR extractant while also producing an ISPR extractant. Theorganic phase 36 can be separated from the aqueous phase 34 of thebiphasic mixture 39 at vessel 35. In some embodiments, separation of thebiphasic mixture 39 can occur in the fermentation vessel, as shown inthe embodiments described in FIGS. 2 and 3 in which thealcohol-containing organic phase stream 36 exits directly fromfermentation vessel 30. Organic phase 36 can be introduced to separator50 for recovery of product alcohol 54 and optional recycle of recoveredextractant 27 as shown in FIG. 1. The remaining process operations ofthe embodiment of FIG. 4 are identical to FIG. 1 and therefore, will notbe described in detail again.

When wet cake 24 is removed via centrifuge 20, in some embodiments, aportion of the oil from feedstock 12, such as corn oil when thefeedstock is corn, remains in wet cake 24. Wet cake 24 can be washedwith additional water in the centrifuge once aqueous solution 22 hasbeen discharged from the centrifuge 20. Washing wet cake 24 will recoverthe sugar (e.g., oligosaccharides) present in the wet cake and therecovered sugar and water can be recycled to the liquefaction vessel 10.After washing, wet cake 20 can be dried to form Dried Distillers' Grainswith Solubles (DDGS) through any suitable known process. The formationof the DDGS from wet cake 24 formed in centrifuge 20 has severalbenefits. Since the undissolved solids do not go to the fermentationvessel, DDGS does not have trapped extractant and/or product alcoholsuch as butanol, it is not subjected to the conditions of thefermentation vessel, and it does not contact the microorganisms presentin the fermentation vessel. All these benefits make it easier to processand sell DDGS, for example, as animal feed. In some embodiments, oil 26is not discharged separately from wet cake 24, but rather oil 26 isincluded as part of wet cake 24 and is ultimately present in the DDGS.In such instances, the oil can be separated from the DDGS and convertedto an ISPR extractant 29 for subsequent use in the same or differentalcohol fermentation process. Methods and systems for removingundissolved solids from feedstock 16 via centrifugation are described indetail in co-pending, commonly owned U.S. Patent Application No.61/356,290, filed Jun. 18, 2010, which is incorporated herein in itsentirety by reference thereto.

In still other embodiments (not shown), saccharification can occur in aseparate saccharification vessel 60 (see FIG. 2) which is locatedbetween separator 20 and liquefaction vessel 10, as should be apparentto one of skill in the art.

In still other embodiments, as shown, for example, in the embodiment ofFIG. 5, a native oil 26′ is supplied to a vessel 40 to which catalyst 42is also supplied, whereby at least a portion of glycerides in oil 26′are hydrolyzed to form FFA 28′. Catalyst 42 can be subsequentlyinactivated, such as by the application of heat q. A product stream fromvessel 40 containing FFA 28′ and inactive catalyst 42 are thenintroduced into fermentation vessel 30, along with aqueous feedstockstream 22 in which feedstock oil 26, and in some embodiments, theundissolved solids have been previously removed by means of separator 20(see, e.g., the embodiment of FIG. 4). Saccharification enzyme 38 andmicroorganism 32 are also introduced into fermentation vessel 30,whereby a product alcohol is produced by SSF.

Alternatively, oil 26′ and catalyst 42 can be fed directly tofermentation vessel 30 in which oil 26′ is hydrolyzed to FFA 28′ ratherthan using vessel 40. Thereafter, active catalyst 42 can be subjected toheat q and inactivated while in the fermentation vessel beforeinoculation of microorganism 32. Alternatively, FFA 28′ and activecatalyst 42 can be fed to fermentation vessel 30 from vessel 40, andactive catalyst 42 can thereafter be subjected to heat q and inactivatedwhile in the fermentation vessel before inoculation of microorganism 32.In such embodiments, feedstock slurry 16 including oil 26, rather thanstream 22 in which oil 26 was removed, can be fed to fermentation vessel30 and contacted with active catalyst 42. Active catalyst 42 cantherefore be used to hydrolyze oil 26 into FFA 28, thereby reducing theloss and/or degradation of the partition coefficient of the extractantover time that is attributable to the presence of the oil in thefermentation vessel.

In some embodiments, the system and processes of FIG. 5 can be modifiedsuch that simultaneous saccharification and fermentation in fermentationvessel 30 is replaced with a separate saccharification vessel 60 priorto fermentation vessel 30, as should be apparent to one of skill in theart (see, e.g., the embodiment of FIG. 2).

In some embodiments, native oil 26′ can be tallow, corn, canola,capric/caprylic triglycerides, castor, coconut, cottonseed, fish,jojoba, lard, linseed, neetsfoot, oiticica, palm, peanut, rapeseed,rice, safflower, soya, sunflower, tung, jatropha, vegetable oil blends,and mixtures thereof. In some embodiments, native oil 26′ is a mixtureof two or more native oils, for example, a mixture of palm and soybeanoils. In some embodiments, native oil 26′ is a plant-derived oil. Insome embodiments, the plant-derived oil can be, though not necessarily,derived from biomass that can be used in a fermentation process. Thebiomass can be the same or different source from which feedstock 12(shown in FIG. 5 as stream 22) is obtained. Thus, for example, in someembodiments, oil 26′ can be derived from corn, whereas feedstock 12 canbe cane. For example, in some embodiments, oil 26′ can be derived fromcorn, and the biomass source of feedstock 12 is also corn. Any possiblecombination of different biomass sources for oil 26′ versus feedstock 12can be used, as should be apparent to one of skill in the art.

FFA 28′ can serve as an ISPR extractant 28′ to form a two-phase mixtureincluding an aqueous phase and an organic phase, with the productalcohol produced in the fermentation medium preferentially partitioninginto the organic phase constituted by ISPR extractant 28′. In someembodiments, one or more additional ISPR extractants 29 can beintroduced into fermentation vessel 30 as described above with referenceto FIG. 1. The organic phase 36 can be separated from the aqueous phase34 of the biphasic mixture 39 at vessel 35. In some embodiments,separation of the biphasic mixture 39 can occur in the fermentationvessel, as shown in the embodiments described in FIGS. 2 and 3 in whichthe alcohol-containing organic phase stream 36 exits directly fromfermentation vessel 30. Organic phase 36 can be introduced in separator50 for recovery of product alcohol 54 and optional recycle of recoveredextractant 27 as shown in FIG. 1. The remaining process operations ofthe embodiment of FIG. 5 are identical to FIG. 1 and therefore, will notbe described in detail again.

In some embodiments of the present invention, biomass oil present infeedstock 12 can be converted to FFA 28 at a step following alcoholicfermentation. FFA 28 can then be introduced as ISPR extractant 28 in thefermentation vessel. For example, in the embodiment of FIG. 6, feedstock12 is liquefied to produced feedstock slurry 16 which includes oil 26derived from the feedstock. Feedstock slurry 16 can also includeundissolved solids from the feedstock. Alternatively, the undissolvedsolids can be separated from slurry 16 via a separator, such as acentrifuge (not shown). Feedstock slurry 16 containing oil 26 isintroduced directly to fermentation vessel 30 containing a fermentationbroth including saccharification enzyme 38 and microorganism 32. Aproduct alcohol is produced by SSF in fermentation vessel 30.Alternatively, in some embodiments, the process can be modified toinclude a separate saccharification vessel as discussed in connectionwith FIG. 2.

ISPR extractant 29 is introduced to fermentation vessel 30 to form abiphasic mixture, and the product alcohol is removed by partitioninginto the organic phase of the ISPR extractant 29. Oil 26 also partitionsinto the organic phase. Separation of the biphasic mixture occurs infermentation vessel 30, whereby alcohol-containing organic phase stream36 and aqueous phase stream 34 exit directly from fermentation vessel30. Alternatively, separation of the biphasic mixture can be conductedin a separate vessel 35 as provided in the embodiments of FIG. 1.Organic phase stream 36 including oil 26 is introduced into separator 50to recover product alcohol 54 from extractant 29. The resultingalcohol-lean extractant 27 includes recovered extractant 29 and oil 26.Extractant 27 is contacted with catalyst 42, whereby at least a portionof glycerides in oil 26 are hydrolyzed to form FFA 28. Heat q can thenbe applied to extractant 27 including FFA 28 so as to inactivatecatalyst 42 before being recycled back into fermentation vessel 30. Suchrecycled extractant stream 27 can be a separate stream or a combinedstream with fresh, make-up extractant stream 29. The subsequentwithdrawal of alcohol-containing organic phase 36 from fermentationvessel 30 can then include FFA 28 and ISPR extractant 29 (as freshextractant 29 and recycled extractant 27), in addition to the productalcohol and additional oil 26 from newly introduced feedstock slurry 16.Organic phase 36 can then be treated to recover the product alcohol, andrecycled back into fermentation vessel 30 after contacting with catalyst42 for hydrolysis of additional oil 26, in the same manner as justdescribed. In some embodiments, use of make-up ISPR extractant 29 can bephased out as the fermentation process is operated over time because theprocess itself can produce FFA 28 as a make-up ISPR extractant forextracting the product alcohol. Thus, the ISPR extractant can be thestream of recycled extractant 27 with FFA 28.

Thus, FIGS. 1-5 provide various non-limiting embodiments of methods andsystems involving fermentation processes and FFAs 28 produced fromcatalytic hydrolysis of biomass derived oil 26, and FFAs 28′ producedfrom catalytic hydrolysis of native oil 26′ such as plant-derived oilthat can be used as ISPR extractants 28 and 28′ to remove productalcohol in extractive fermentation.

In some embodiments, including any of the aforementioned embodimentsdescribed with reference to FIGS. 1-6, the fermentation broth infermentation vessel 30 includes at least one recombinant microorganism32 which is genetically modified (that is, genetically engineered) toproduce butanol via a biosynthetic pathway from at least one fermentablecarbon source. In particular, recombinant microorganisms can be grown ina fermentation broth which contains suitable carbon substrates.Additional carbon substrates may include, but are not limited to,monosaccharides such as fructose; oligosaccharides such as lactose,maltose, or sucrose; polysaccharides such as starch or cellulose; ormixtures thereof, and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Other carbon substrates may include ethanol, lactate,succinate, or glycerol.

Additionally the carbon substrate may also be one-carbon substrates suchas carbon dioxide or methanol for which metabolic conversion into keybiochemical intermediates has been demonstrated. In addition to one andtwo carbon substrates, methylotrophic organisms are also known toutilize a number of other carbon containing compounds such asmethylamine, glucosamine, and a variety of amino acids for metabolicactivity. For example, methylotrophic yeasts are known to utilize thecarbon from methylamine to form trehalose or glycerol (Bellion, et al.,Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32, Editor(s):Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).Similarly, various species of Candida will metabolize alanine or oleicacid (Sulter, et al., Arch. Microbiol. 153:485-489, 1990). Hence it iscontemplated that the source of carbon utilized in the present inventionmay encompass a wide variety of carbon containing substrates and willonly be limited by the choice of organism.

Although it is contemplated that all of the above mentioned carbonsubstrates and mixtures thereof are suitable, in some embodiments, thecarbon substrates are glucose, fructose, and sucrose, or mixtures ofthese with C5 sugars such as xylose and/or arabinose for yeasts cellsmodified to use C5 sugars. Sucrose may be derived from renewable sugarsources such as sugar cane, sugar beets, cassaya, sweet sorghum, andmixtures thereof. Glucose and dextrose may be derived from renewablegrain sources through saccharification of starch based feedstocksincluding grains such as corn, wheat, rye, barley, oats, and mixturesthereof. In addition, fermentable sugars may be derived from renewablecellulosic or lignocellulosic biomass through processes of pretreatmentand saccharification, as described in, for example, in U.S. PatentApplication Publication No. 2007/0031918 A1, which is hereinincorporated by reference. In addition to an appropriate carbon source(from aqueous stream 22), fermentation broth must contain suitableminerals, salts, cofactors, buffers and other components, known to thoseskilled in the art, suitable for the growth of the cultures andpromotion of an enzymatic pathway comprising a dihydroxyacid dehydratase(DHAD).

Recombinant microorganisms that produce butanol via a biosyntheticpathway can include a member of the genera Clostridium, Zymomonas,Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella,Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus,Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium,Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia,Candida, Hansenula, or Saccharomyces. In one embodiment, recombinantmicroorganisms can be selected from the group consisting of Escherichiacoli, Lactobacillus plantarum, and Saccharomyces cerevisiae. In oneembodiment, the recombinant microorganism is a crabtree-positive yeastselected from Saccharomyces, Zygosaccharomyces, Schizosaccharomyces,Dekkera, Torulopsis, Brettanomyces, and some species of Candida. Speciesof crabtree-positive yeast include, but are not limited to,Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomycespombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomycesparadoxus, Zygosaccharomyces rouxii, and Candida glabrata. For example,the production of butanol utilizing fermentation with a microorganism,as well as which microorganisms produce butanol, is known and isdisclosed, for example, in U.S. Patent Application Publication No.2009/0305370, herein incorporated by reference. In some embodiments,microorganisms comprise a butanol biosynthetic pathway. Suitableisobutanol biosynthetic pathways are known in the art (see, e.g., U.S.Patent Application Publication No. 2007/0092957, herein incorporated byreference). In some embodiments, at least one, at least two, at leastthree, or at least four polypeptides catalyzing substrate to productconversions of a pathway are encoded by heterologous polynucleotides inthe microorganism. In some embodiments, all polypeptides catalyzingsubstrate to product conversions of a pathway are encoded byheterologous polynucleotides in the microorganism. In some embodiments,the microorganism comprises a reduction or elimination of pyruvatedecarboxylase activity. Microorganisms substantially free of pyruvatedecarboxylase activity are described in U.S. Patent ApplicationPublication No. 2009/0305363, herein incorporated by reference.

Construction of certain strains, including those used in the Examples,is provided herein.

Construction of Saccharomyces cerevisiae Strain BP1083 (“NGCI-070”)

The strain BP1064 was derived from CEN.PK 113-7D (CBS 8340;Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre,Netherlands) and contains deletions of the following genes: URA3, HIS3,PDC1, PDC5, PDC6, and GPD2. BP1064 was transformed with plasmids pYZ090(SEQ ID NO: 1, described in U.S. Provisional Application Ser. No.61/246,844) and pLH468 (SEQ ID NO: 2) to create strain NGCI-070 (BP1083,PNY1504).

Deletions, which completely removed the entire coding sequence, werecreated by homologous recombination with PCR fragments containingregions of homology upstream and downstream of the target gene andeither a G418 resistance marker or URA3 gene for selection oftransformants. The G418 resistance marker, flanked by loxP sites, wasremoved using Cre recombinase. The URA3 gene was removed by homologousrecombination to create a scarless deletion or if flanked by loxP sites,was removed using Cre recombinase.

The scarless deletion procedure was adapted from Akada, et al., (Yeast23:399-405, 2006). In general, the PCR cassette for each scarlessdeletion was made by combining four fragments, A-B-U-C, by overlappingPCR. The PCR cassette contained a selectable/counter-selectable marker,URA3 (Fragment U), consisting of the native CEN.PK 113-7D URA3 gene,along with the promoter (250 bp upstream of the URA3 gene) andterminator (150 bp downstream of the URA3 gene). Fragments A and C, each500 bp long, corresponded to the 500 bp immediately upstream of thetarget gene (Fragment A) and the 3′ 500 bp of the target gene (FragmentC). Fragments A and C were used for integration of the cassette into thechromosome by homologous recombination. Fragment B (500 bp long)corresponded to the 500 bp immediately downstream of the target gene andwas used for excision of the URA3 marker and Fragment C from thechromosome by homologous recombination, as a direct repeat of thesequence corresponding to Fragment B was created upon integration of thecassette into the chromosome. Using the PCR product ABUC cassette, theURA3 marker was first integrated into and then excised from thechromosome by homologous recombination. The initial integration deletedthe gene, excluding the 3′ 500 bp. Upon excision, the 3′ 500 bp regionof the gene was also deleted. For integration of genes using thismethod, the gene to be integrated was included in the PCR cassettebetween fragments A and B.

URA3 Deletion

To delete the endogenous URA3 coding region, a ura3::loxP-kanMX-IoxPcassette was PCR-amplified from pLA54 template DNA (SEQ ID NO: 3). pLA54contains the K. lactis TEF1 promoter and kanMX marker, and is flanked byloxP sites to allow recombination with Cre recombinase and removal ofthe marker. PCR was done using Phusion® DNA polymerase (New EnglandBioLabs Inc., Ipswich, Mass.) and primers BK505 and BK506 (SEQ ID NOs: 4and 5). The URA3 portion of each primer was derived from the 5′ regionupstream of the URA3 promoter and 3′ region downstream of the codingregion such that integration of the loxP-kanMX-IoxP marker resulted inreplacement of the URA3 coding region. The PCR product was transformedinto CEN.PK 113-7D using standard genetic techniques (Methods in YeastGenetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., pp. 201-202) and transformants were selected on YPD containingG418 (100 μg/mL) at 30° C. Transformants were screened to verify correctintegration by PCR using primers LA468 and LA492 (SEQ ID NOs: 6 and 7)and designated CEN.PK 113-7D Δura3::kanMX.

HIS3 Deletion

The four fragments for the PCR cassette for the scarless HIS3 deletionwere amplified using Phusion® High Fidelity PCR Master Mix (New EnglandBioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template,prepared with a Gentra® Puregene® Yeast/Bact, kit (Qiagen, Valencia,Calif.). HIS3 Fragment A was amplified with primer oBP452 (SEQ ID NO:14) and primer oBP453 (SEQ ID NO: 15) containing a 5′ tail with homologyto the 5′ end of HIS3 Fragment B. HIS3 Fragment B was amplified withprimer oBP454 (SEQ ID NO: 16) containing a 5′ tail with homology to the3′ end of HIS3 Fragment A, and primer oBP455 (SEQ ID NO: 17) containinga 5′ tail with homology to the 5′ end of HIS3 Fragment U. HIS3 FragmentU was amplified with primer oBP456 (SEQ ID NO: 18) containing a 5′ tailwith homology to the 3′ end of HIS3 Fragment B, and primer oBP457 (SEQID NO: 19) containing a 5′ tail with homology to the 5′ end of HIS3Fragment C. HIS3 Fragment C was amplified with primer oBP458 (SEQ ID NO:20) containing a 5′ tail with homology to the 3′ end of HIS3 Fragment U,and primer oBP459 (SEQ ID NO: 21). PCR products were purified with a PCRPurification kit (Qiagen, Valencia, Calif.). HIS3 Fragment AB wascreated by overlapping PCR by mixing HIS3 Fragment A and HIS3 Fragment Band amplifying with primers oBP452 (SEQ ID NO: 14) and oBP455 (SEQ IDNO: 17). HIS3 Fragment UC was created by overlapping PCR by mixing HIS3Fragment U and HIS3 Fragment C and amplifying with primers oBP456 (SEQID NO: 18) and oBP459 (SEQ ID NO: 21). The resulting PCR products werepurified on an agarose gel followed by a Gel Extraction kit (Qiagen,Valencia, Calif.). The HIS3 ABUC cassette was created by overlapping PCRby mixing HIS3 Fragment AB and HIS3 Fragment UC and amplifying withprimers oBP452 (SEQ ID NO: 14) and oBP459 (SEQ ID NO: 21). The PCRproduct was purified with a PCR Purification kit (Qiagen, Valencia,Calif.).

Competent cells of CEN.PK 113-7D Δura3::kanMX were made and transformedwith the HIS3 ABUC PCR cassette using a Frozen-EZ Yeast TransformationII™ kit (Zymo Research Corporation, Irvine, Calif.). Transformationmixtures were plated on synthetic complete media lacking uracilsupplemented with 2% glucose at 30° C. Transformants with a his3knockout were screened for by PCR with primers oBP460 (SEQ ID NO: 22)and oBP461 (SEQ ID NO: 23) using genomic DNA prepared with a Gentra®Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). A correcttransformant was selected as strain CEN.PK 113-7D Δura3::kanMXΔhis3::URA3.

KanMX Marker Removal from the Δura3 Site and URA3 Marker Removal fromthe Δhis3 Site

The KanMX marker was removed by transforming CEN.PK 113-7D Δura3::kanMXΔhis3::URA3 with pRS423::PGAL1-cre (SEQ ID NO: 66, described in U.S.Provisional Application No. 61/290,639) using a Frozen-EZ YeastTransformation II™ kit (Zymo Research Corporation, Irvine, Calif.) andplating on synthetic complete medium lacking histidine and uracilsupplemented with 2% glucose at 30° C. Transformants were grown in YPsupplemented with 1% galactose at 30° C. for ˜6 hours to induce the Crerecombinase and KanMX marker excision and plated onto YPD (2% glucose)plates at 30° C. for recovery. An isolate was grown overnight in YPD andplated on synthetic complete medium containing 5-fluoro-orotic acid(5-FOA, 0.1%) at 30° C. to select for isolates that lost the URA3marker. 5-FOA resistant isolates were grown in and plated on YPD forremoval of the pRS423::PGAL1-cre plasmid. Isolates were checked for lossof the KanMX marker, URA3 marker, and pRS423::PGAL1-cre plasmid byassaying growth on YPD+G418 plates, synthetic complete medium lackinguracil plates, and synthetic complete medium lacking histidine plates. Acorrect isolate that was sensitive to G418 and auxotrophic for uraciland histidine was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 anddesignated as BP857. The deletions and marker removal were confirmed byPCR and sequencing with primers oBP450 (SEQ ID NO: 24) and oBP451 (SEQID NO: 25) for Δura3 and primers oBP460 (SEQ ID NO: 22) and oBP461 (SEQID NO: 23) for Δhis3 using genomic DNA prepared with a Gentra® Puregene®Yeast/Bact. kit (Qiagen, Valencia, Calif.).

PDC6 Deletion

The four fragments for the PCR cassette for the scarless PDC6 deletionwere amplified using Phusion® High Fidelity PCR Master Mix (New EnglandBioLabs Inc., Ipswich, Mass.) and CEN.PK 113-7D genomic DNA as template,prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia,Calif.). PDC6 Fragment A was amplified with primer oBP440 (SEQ ID NO:26) and primer oBP441 (SEQ ID NO: 27) containing a 5′ tail with homologyto the 5′ end of PDC6 Fragment B. PDC6 Fragment B was amplified withprimer oBP442 (SEQ ID NO: 28), containing a 5′ tail with homology to the3′ end of PDC6 Fragment A, and primer oBP443 (SEQ ID NO: 29) containinga 5′ tail with homology to the 5′ end of PDC6 Fragment U. PDC6 FragmentU was amplified with primer oBP444 (SEQ ID NO: 30) containing a 5′ tailwith homology to the 3′ end of PDC6 Fragment B, and primer oBP445 (SEQID NO: 31) containing a 5′ tail with homology to the 5′ end of PDC6Fragment C. PDC6 Fragment C was amplified with primer oBP446 (SEQ ID NO:32) containing a 5′ tail with homology to the 3′ end of PDC6 Fragment U,and primer oBP447 (SEQ ID NO: 33). PCR products were purified with a PCRPurification kit (Qiagen, Valencia, Calif.). PDC6 Fragment AB wascreated by overlapping PCR by mixing PDC6 Fragment A and PDC6 Fragment Band amplifying with primers oBP440 (SEQ ID NO: 26) and oBP443 (SEQ IDNO: 29). PDC6 Fragment UC was created by overlapping PCR by mixing PDC6Fragment U and PDC6 Fragment C and amplifying with primers oBP444 (SEQID NO: 30) and oBP447 (SEQ ID NO: 33). The resulting PCR products werepurified on an agarose gel followed by a Gel Extraction kit (Qiagen,Valencia, Calif.). The PDC6 ABUC cassette was created by overlapping PCRby mixing PDC6 Fragment AB and PDC6 Fragment UC and amplifying withprimers oBP440 (SEQ ID NO: 26) and oBP447 (SEQ ID NO: 33). The PCRproduct was purified with a PCR Purification kit (Qiagen, Valencia,Calif.).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 were made andtransformed with the PDC6 ABUC PCR cassette using a Frozen-EZ YeastTransformation II™ kit (Zymo Research Corporation, Irvine, Calif.).Transformation mixtures were plated on synthetic complete media lackinguracil supplemented with 2% glucose at 30° C. Transformants with a pdc6knockout were screened for by PCR with primers oBP448 (SEQ ID NO: 34)and oBP449 (SEQ ID NO: 35) using genomic DNA prepared with a Gentra®Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). A correcttransformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3Δpdc6::URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6::URA3 was grown overnight in YPDand plated on synthetic complete medium containing 5-fluoro-orotic acid(0.1%) at 30° C. to select for isolates that lost the URA3 marker. Thedeletion and marker removal were confirmed by PCR and sequencing withprimers oBP448 (SEQ ID NO: 34) and oBP449 (SEQ ID NO: 35) using genomicDNA prepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia,Calif.). The absence of the PDC6 gene from the isolate was demonstratedby a negative PCR result using primers specific for the coding sequenceof PDC6, oBP554 (SEQ ID NO: 36) and oBP555 (SEQ ID NO: 37). The correctisolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 anddesignated as BP891.

PDC1 Deletion ilvDSm Integration

The PDC1 gene was deleted and replaced with the ilvD coding region fromStreptococcus mutans ATCC No. 700610. The A fragment followed by theilvD coding region from Streptococcus mutans for the PCR cassette forthe PDC1 deletion-ilvDSm integration was amplified using Phusion® HighFidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) andNYLA83 (described herein and in U.S. Provisional Application No.61/246,709) genomic DNA as template, prepared with a Gentra® Puregene®Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC1 Fragment A-ilvDSm (SEQID NO: 141) was amplified with primer oBP513 (SEQ ID NO: 38) and primeroBP515 (SEQ ID NO: 39) containing a 5′ tail with homology to the 5′ endof PDC1 Fragment B. The B, U, and C fragments for the PCR cassette forthe PDC1 deletion-ilvDSm integration were amplified using Phusion® HighFidelity PCR Master Mix (New England BioLabs Inc., Ipswich, Mass.) andCEN.PK 113-7D genomic DNA as template, prepared with a Gentra® Puregene®Yeast/Bact. kit (Qiagen, Valencia, Calif.). PDC1 Fragment B wasamplified with primer oBP516 (SEQ ID NO: 40) containing a 5′ tail withhomology to the 3′ end of PDC1 Fragment A-ilvDSm, and primer oBP517 (SEQID NO: 41) containing a 5′ tail with homology to the 5′ end of PDC1Fragment U. PDC1 Fragment U was amplified with primer oBP518 (SEQ ID NO:42) containing a 5′ tail with homology to the 3′ end of PDC1 Fragment B,and primer oBP519 (SEQ ID NO: 43) containing a 5′ tail with homology tothe 5′ end of PDC1 Fragment C. PDC1 Fragment C was amplified with primeroBP520 (SEQ ID NO: 44), containing a 5′ tail with homology to the 3′ endof PDC1 Fragment U, and primer oBP521 (SEQ ID NO: 45). PCR products werepurified with a PCR Purification kit (Qiagen, Valencia, Calif. PDC1Fragment A-ilvDSm-B was created by overlapping PCR by mixing PDC1Fragment A-ilvDSm and PDC1 Fragment B and amplifying with primers oBP513(SEQ ID NO: 38) and oBP517 (SEQ ID NO: 41). PDC1 Fragment UC was createdby overlapping PCR by mixing PDC1 Fragment U and PDC1 Fragment C andamplifying with primers oBP518 (SEQ ID NO: 42) and oBP521 (SEQ ID NO:45). The resulting PCR products were purified on an agarose gel followedby a Gel Extraction kit (Qiagen, Valencia, Calif.). The PDC1A-ilvDSm-BUC cassette (SEQ ID NO: 142) was created by overlapping PCR bymixing PDC1 Fragment A-ilvDSm-B and PDC1 Fragment UC and amplifying withprimers oBP513 (SEQ ID NO: 38) and oBP521 (SEQ ID NO: 45). The PCRproduct was purified with a PCR Purification kit (Qiagen, Valencia,Calif.).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 were made andtransformed with the PDC1 A-ilvDSm-BUC PCR cassette using a Frozen-EZYeast Transformation II™ kit (Zymo Research Corporation, Irvine,Calif.). Transformation mixtures were plated on synthetic complete medialacking uracil supplemented with 2% glucose at 30° C. Transformants witha pdc1 knockout ilvDSm integration were screened for by PCR with primersoBP511 (SEQ ID NO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNAprepared with a Gentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia,Calif.). The absence of the PDC1 gene from the isolate was demonstratedby a negative PCR result using primers specific for the coding sequenceof PDC1, oBP550 (SEQ ID NO: 48) and oBP551 (SEQ ID NO: 49). A correcttransformant was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3Δpdc6 Δpdc1::ilvDSm-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm-URA3 was grownovernight in YPD and plated on synthetic complete medium containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC1, integration of ilvDSm, and markerremoval were confirmed by PCR and sequencing with primers oBP511 (SEQ IDNO: 46) and oBP512 (SEQ ID NO: 47) using genomic DNA prepared with aGentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). Thecorrect isolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3Δpdc6 Δpdc1::ilvDSm and designated as BP907.

PDC5 Deletion sadB Integration

The PDC5 gene was deleted and replaced with the sadB coding region fromAchromobacter xylosoxidans. A segment of the PCR cassette for the PDC5deletion-sadB integration was first cloned into plasmid pUC19-URA3MCS.

pUC19-URA3MCS is pUC19 based and contains the sequence of the URA3 genefrom Saccaromyces cerevisiae situated within a multiple cloning site(MCS). pUC19 contains the pMB1 replicon and a gene coding forbeta-lactamase for replication and selection in Escherichia coli. Inaddition to the coding sequence for URA3, the sequences from upstreamand downstream of this gene were included for expression of the URA3gene in yeast. The vector can be used for cloning purposes and can beused as a yeast integration vector.

The DNA encompassing the URA3 coding region along with 250 bp upstreamand 150 bp downstream of the URA3 coding region from Saccaromycescerevisiae CEN.PK 113-7D genomic DNA was amplified with primers oBP438(SEQ ID NO: 12) containing BamHI, Ascl, Pmel, and FseI restrictionsites, and oBP439 (SEQ ID NO: 13) containing XbaI, PacI, and NotIrestriction sites, using Phusion® High Fidelity PCR Master Mix (NewEngland BioLabs Inc., Ipswich, Mass.). Genomic DNA was prepared using aGentra® Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The PCRproduct and pUC19 (SEQ ID NO: 143) were ligated with T4 DNA ligase afterdigestion with BamHI and XbaI to create vector pUC19-URA3MCS. The vectorwas confirmed by PCR and sequencing with primers oBP264 (SEQ ID NO: 10)and oBP265 (SEQ ID NO: 11).

The coding sequence of sadB and PDC5 Fragment B were cloned intopUC19-URA3MCS to create the sadB-BU portion of the PDC5 A-sadB-BUC PCRcassette. The coding sequence of sadB was amplified using pLH468-sadB(SEQ ID NO: 67) as template with primer oBP530 (SEQ ID NO: 50)containing an AscI restriction site, and primer oBP531 (SEQ ID NO: 51)containing a 5′ tail with homology to the 5′ end of PDC5 Fragment B.PDC5 Fragment B was amplified with primer oBP532 (SEQ ID NO: 52)containing a 5′ tail with homology to the 3′ end of sadB, and primeroBP533 (SEQ ID NO: 53) containing a PmeI restriction site. PCR productswere purified with a PCR Purification kit (Qiagen, Valencia, Calif.).sadB-PDC5 Fragment B was created by overlapping PCR by mixing the sadBand PDC5 Fragment B PCR products and amplifying with primers oBP530 (SEQID NO: 50) and oBP533 (SEQ ID NO: 53). The resulting PCR product wasdigested with AscI and PmeI and ligated with T4 DNA ligase into thecorresponding sites of pUC19-URA3MCS after digestion with theappropriate enzymes. The resulting plasmid was used as a template foramplification of sadB-Fragment B-Fragment U using primers oBP536 (SEQ IDNO: 54) and oBP546 (SEQ ID NO: 55) containing a 5′ tail with homology tothe 5′ end of PDC5 Fragment C. PDC5 Fragment C was amplified with primeroBP547 (SEQ ID NO: 56) containing a 5′ tail with homology to the 3′ endof PDC5 sadB-Fragment B-Fragment U, and primer oBP539 (SEQ ID NO: 57).PCR products were purified with a PCR Purification kit (Qiagen,Valencia, Calif.). PDC5 sadB-Fragment B-Fragment U-Fragment C wascreated by overlapping PCR by mixing PDC5 sadB-Fragment B-Fragment U andPDC5 Fragment C and amplifying with primers oBP536 (SEQ ID NO: 54) andoBP539 (SEQ ID NO: 57). The resulting PCR product was purified on anagarose gel followed by a Gel Extraction kit (Qiagen, Valencia, Calif.).The PDC5 A-sadB-BUC cassette (SEQ ID NO: 144) was created by amplifyingPDC5 sadB-Fragment B-Fragment U-Fragment C with primers oBP542 (SEQ IDNO: 58) containing a 5′ tail with homology to the 50 nucleotidesimmediately upstream of the native PDC5 coding sequence, and oBP539 (SEQID NO: 57). The PCR product was purified with a PCR Purification kit(Qiagen, Valencia, Calif.).

Competent cells of CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSmwere made and transformed with the PDC5 A-sadB-BUC PCR cassette using aFrozen-EZ Yeast Transformation II™ kit (Zymo Research Corporation,Irvine, Calif.). Transformation mixtures were plated on syntheticcomplete media lacking uracil supplemented with 1% ethanol (no glucose)at 30° C. Transformants with a pdc5 knockout sadB integration werescreened for by PCR with primers oBP540 (SEQ ID NO: 59) and oBP541 (SEQID NO: 60) using genomic DNA prepared with a Gentra® Puregene®Yeast/Bact. kit (Qiagen, Valencia, Calif.). The absence of the PDC5 genefrom the isolate was demonstrated by a negative PCR result using primersspecific for the coding sequence of PDC5, oBP552 (SEQ ID NO: 61) andoBP553 (SEQ ID NO: 62). A correct transformant was selected as strainCEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3.

CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6 Δpdc1::ilvDSm Δpdc5::sadB-URA3 wasgrown overnight in YPE (1% ethanol) and plated on synthetic completemedium supplemented with ethanol (no glucose) and containing5-fluoro-orotic acid (0.1%) at 30° C. to select for isolates that lostthe URA3 marker. The deletion of PDC5, integration of sadB, and markerremoval were confirmed by PCR with primers oBP540 (SEQ ID NO: 59) andoBP541 (SEQ ID NO: 60) using genomic DNA prepared with a Gentra®Puregene® Yeast/Bact. kit (Qiagen, Valencia, Calif.). The correctisolate was selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6Δpdc1::ilvDSm Δpdc5::sadB and designated as BP913.

GPD2 Deletion

To delete the endogenous GPD2 coding region, a gpd2::loxP-URA3-loxPcassette (SEQ ID NO: 145) was PCR-amplified using loxP-URA3-loxP (SEQ IDNO: 68) as template DNA. loxP-URA3-loxP contains the URA3 marker from(ATCC No. 77107) flanked by loxP recombinase sites. PCR was done usingPhusion® DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) andprimers LA512 and LA513 (SEQ ID NOs: 8 and 9). The GPD2 portion of eachprimer was derived from the 5′ region upstream of the GPD2 coding regionand 3′ region downstream of the coding region such that integration ofthe loxP-URA3-loxP marker resulted in replacement of the GPD2 codingregion. The PCR product was transformed into BP913 and transformantswere selected on synthetic complete media lacking uracil supplementedwith 1% ethanol (no glucose). Transformants were screened to verifycorrect integration by PCR using primers oBP582 and AA270 (SEQ ID NOs:63 and 64).

The URA3 marker was recycled by transformation with pRS423::PGAL1-cre(SEQ ID NO: 66) and plating on synthetic complete media lackinghistidine supplemented with 1% ethanol at 30° C. Transformants werestreaked on synthetic complete medium supplemented with 1% ethanol andcontaining 5-fluoro-orotic acid (0.1%) and incubated at 30° C. to selectfor isolates that lost the URA3 marker. 5-FOA resistant isolates weregrown in YPE (1% ethanol) for removal of the pRS423::PGAL1-cre plasmid.The deletion and marker removal were confirmed by PCR with primersoBP582 (SEQ ID NO: 63) and oBP591 (SEQ ID NO: 65). The correct isolatewas selected as strain CEN.PK 113-7D Δura3::loxP Δhis3 Δpdc6Δpdc1::ilvDSm Δpdc5::sadB Δgpd2::loxP and designated as PNY1503(BP1064).

BP1064 was transformed with plasmids pYZ090 (SEQ ID NO: 1) and pLH468(SEQ ID NO: 2) to create strain NGCI-070 (BP1083; PNY1504).

Construction of Strains NYLA74, NYLA83, and NYLA84

Insertion-inactivation of endogenous PDC1 and PDC6 genes of S.cerevisiae. PDC1, PDC5, and PDC6 genes encode the three major isozymesof pyruvate decarboxylase is described as follows:

Construction of pRS425::GPM-sadB

A DNA fragment encoding a butanol dehydrogenase (SEQ ID NO: 70) fromAchromobacter xylosoxidans (disclosed in U.S. Patent ApplicationPublication No. 2009/0269823) was cloned. The coding region of this genecalled sadB for secondary alcohol dehydrogenase (SEQ ID NO: 69) wasamplified using standard conditions from A. xylosoxidans genomic DNA,prepared using a Gentra® Puregene® kit (Qiagen, Valencia, Calif.)following the recommended protocol for gram negative organisms usingforward and reverse primers N473 and N469 (SEQ ID NOs: 74 and 75),respectively. The PCR product was TOPO®-Blunt cloned into pCR®4 BLUNT(Invitrogen™, Carlsbad, Calif.) to produce pCR4Blunt::sadB, which wastransformed into E. coli Mach-1 cells. Plasmid was subsequently isolatedfrom four clones, and the sequence verified.

The sadB coding region was PCR amplified from pCR4Blunt::sadB. PCRprimers contained additional 5′ sequences that would overlap with theyeast GPM1 promoter and the ADH1 terminator (N583 and N584, provided asSEQ ID NOs: 76 and 77). The PCR product was then cloned using “gaprepair” methodology in Saccharomyces cerevisiae (Ma, et al., Gene58:201-216, 1987) as follows. The yeast-E. coli shuttle vectorpRS425::GPM::kivD::ADH which contains the GPM1 promoter (SEQ ID NO: 72),kivD coding region from Lactococcus lactis (SEQ ID NO: 71), and ADH1terminator (SEQ ID NO: 73) (described in U.S. Patent ApplicationPublication No. 2007/0092957 A1, Example 17) was digested with BbvCl andPacI restriction enzymes to release the kivD coding region.Approximately 1 μg of the remaining vector fragment was transformed intoS. cerevisiae strain BY4741 along with 1 μg of sadB PCR product.Transformants were selected on synthetic complete medium lackingleucine. The proper recombination event, generating pRS425::GPM-sadB,was confirmed by PCR using primers N142 and N459 (SEQ ID NOs: 108 and109).

Construction of pdc6:: PGPM1-sadB Integration Cassette and PDC6Deletion:

A pdc6::PGPM1-sadB-ADH1t-URA3r integration cassette was made by joiningthe GPM-sadB-ADHt segment (SEQ ID NO: 79) from pRS425::GPM-sadB (SEQ IDNO: 78) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO:80)contains the URA3 marker from pRS426 (ATCC No. 77107) flanked by 75 bphomologous repeat sequences to allow homologous recombination in vivoand removal of the URA3 marker. The two DNA segments were joined by SOEPCR (as described by Horton, et al., Gene 77:61-68, 1989) using astemplate pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion®DNA polymerase (New England BioLabs Inc., Ipswich, Mass.) and primers114117-11A through 114117-11D (SEQ ID NOs: 81, 82, 83, and 84), and114117-13A and 114117-13B (SEQ ID NOs: 85 and 86).

The outer primers for the SOE PCR (114117-13A and 114117-13B) contained5′ and 3′ ˜50 bp regions homologous to regions upstream and downstreamof the PDC6 promoter and terminator, respectively. The completedcassette PCR fragment was transformed into BY4700 (ATCC No. 200866) andtransformants were maintained on synthetic complete media lacking uraciland supplemented with 2% glucose at 30° C. using standard genetictechniques (Methods in Yeast Genetics, 2005, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformantswere screened by PCR using primers 112590-34G and 112590-34H (SEQ IDNOs: 87 and 88), and 112590-34F and 112590-49E (SEQ ID NOs: 89 and 90)to verify integration at the PDC6 locus with deletion of the PDC6 codingregion. The URA3r marker was recycled by plating on synthetic completemedia supplemented with 2% glucose and 5-FOA at 30° C. followingstandard protocols. Marker removal was confirmed by patching coloniesfrom the 5-FOA plates onto SD-URA media to verify the absence of growth.The resulting identified strain has the genotype: BY4700pdc6::PGPM1-sadB-ADH1t.

Construction of pdc1:: PPDC1-ilvD Integration Cassette and PDC1Deletion:

A pdc1:: PPDC1-ilvD-FBA1t-URA3r integration cassette was made by joiningthe ilvD-FBA1t segment (SEQ ID NO: 91) from pLH468 (SEQ ID NO: 2) to theURA3r gene from pUC19-URA3r by SOE PCR (as described by Horton, et al.,Gene 77:61-68, 1989) using as template pLH468 and pUC19-URA3r plasmidDNAs, with Phusion® DNA polymerase (New England BioLabs Inc., Ipswich,Mass.) and primers 114117-27A through 114117-27D (SEQ ID NOs: 111, 112,113, and 114).

The outer primers for the SOE PCR (114117-27A and 114117-27D) contained5′ and 3′ ˜50 bp regions homologous to regions downstream of the PDC1promoter and downstream of the PDC1 coding sequence. The completedcassette PCR fragment was transformed into BY4700 pdc6::PGPM1-sadB-ADH1tand transformants were maintained on synthetic complete media lackinguracil and supplemented with 2% glucose at 30° C. using standard genetictechniques (Methods in Yeast Genetics, 2005, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformantswere screened by PCR using primers 114117-36D and 135 (SEQ ID NOs: 92and 93), and primers 112590-49E and 112590-30F (SEQ ID NOs: 90 and 94)to verify integration at the PDC1 locus with deletion of the PDC1 codingsequence. The URA3r marker was recycled by plating on synthetic completemedia supplemented with 2% glucose and 5-FOA at 30° C. followingstandard protocols. Marker removal was confirmed by patching coloniesfrom the 5-FOA plates onto SD-URA media to verify the absence of growth.The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6::PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t.

HIS3 Deletion

To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette wasPCR-amplified from URA3r2 template DNA (SEQ ID NO: 95). URA3r2 containsthe URA3 marker from pRS426 (ATCC No. 77107) flanked by 500 bphomologous repeat sequences to allow homologous recombination in vivoand removal of the URA3 marker. PCR was done using Phusion® DNApolymerase (New England BioLabs Inc., Ipswich, Mass.) and primers114117-45A and 114117-45B (SEQ ID NOs: 96 and 97) which generated a ˜2.3kb PCR product. The HIS3 portion of each primer was derived from the 5′region upstream of the HIS3 promoter and 3′ region downstream of thecoding region such that integration of the URA3r2 marker results inreplacement of the HIS3 coding region. The PCR product was transformedinto NYLA67 using standard genetic techniques (Methods in YeastGenetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., pp. 201-202) and transformants were selected on synthetic completemedia lacking uracil and supplemented with 2% glucose at 30° C.Transformants were screened to verify correct integration by replicaplating of transformants onto synthetic complete media lacking histidineand supplemented with 2% glucose at 30° C. The URA3r marker was recycledby plating on synthetic complete media supplemented with 2% glucose and5-FOA at 30° C. following standard protocols. Marker removal wasconfirmed by patching colonies from the 5-FOA plates onto SD-URA mediato verify the absence of growth. The resulting identified strain, calledNYLA73, has the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1::PPDC1-ilvD-FBA1t Δhis3.

Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:

A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134Wchromosomal DNA (ATCC No. 4034091) using Phusion® DNA polymerase (NewEngland BioLabs Inc., Ipswich, Mass.) and primers PDC5::KanMXF andPDC5::KanMXR (SEQ ID NOs: 98 and 99) which generated a ˜2.2 kb PCRproduct. The PDC5 portion of each primer was derived from the 5′ regionupstream of the PDC5 promoter and 3′ region downstream of the codingregion such that integration of the kanMX4 marker results in replacementof the PDC5 coding region. The PCR product was transformed into NYLA73using standard genetic techniques (Methods in Yeast Genetics, 2005, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202)and transformants were selected on YP media supplemented with 1% ethanoland geneticin (200 μg/mL) at 30° C. Transformants were screened by PCRto verify correct integration at the PDC locus with replacement of thePDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 100 and101). The identified correct transformants have the genotype: BY4700pdc6:: PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 pdc5::kanMX4. Thestrain was named NYLA74.

Plasmid vectors pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadBwere transformed into NYLA74 to create a butanediol producing strain(NGCI-047).

Plasmid vectors pLH475-Z4B8 (SEQ ID NO: 140) and pLH468 were transformedinto NYLA74 to create an isobutanol producing strain (NGCI-049).

Deletion of HXK2 (Hexokinase II):

A hxk2::URA3r cassette was PCR-amplified from URA3r2 template (describedabove) using Phusion® DNA polymerase (New England BioLabs Inc., Ipswich,Mass.) and primers 384 and 385 (SEQ ID NOs: 102 and 103) which generateda ˜2.3 kb PCR product. The HXK2 portion of each primer was derived fromthe 5′ region upstream of the HXK2 promoter and 3′ region downstream ofthe coding region such that integration of the URA3r2 marker results inreplacement of the HXK2 coding region. The PCR product was transformedinto NYLA73 using standard genetic techniques (Methods in YeastGenetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., pp. 201-202) and transformants were selected on synthetic completemedia lacking uracil and supplemented with 2% glucose at 30° C.Transformants were screened by PCR to verify correct integration at theHXK2 locus with replacement of the HXK2 coding region using primers N869and N871 (SEQ ID NOs: 104 and 105). The URA3r2 marker was recycled byplating on synthetic complete media supplemented with 2% glucose and5-FOA at 30° C. following standard protocols. Marker removal wasconfirmed by patching colonies from the 5-FOA plates onto SD-URA mediato verify the absence of growth, and by PCR to verify correct markerremoval using primers N946 and N947 (SEQ ID NOs: 106 and 107). Theresulting identified strain named NYLA83 has the genotype: BY4700 pdc6::PGPM1-sadB-ADH1t pdc1:: PPDC1-ilvD-FBA1t Δhis3 Δhxk2.

Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:

A pdc5::kanMX4 cassette was PCR-amplified as described above. The PCRfragment was transformed into NYLA83, and transformants were selectedand screened as described above. The identified correct transformantsnamed NYLA84 have the genotype: BY4700 pdc6:: PGPM1-sadB-ADH1t pdc1::PPDC1-ilvD-FBA1t Δhis3 Δhxk2 pdc5::kanMX4.

Plasmid vectors pLH468 and pLH532 were simultaneously transformed intostrain NYLA84 (BY4700 pdc6::PGPM1-sadB-ADH1t pdc1::PPDC1-ilvD-FBA1tΔhis3 Δhxk2 pdc5::kanMX4) using standard genetic techniques (Methods inYeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.) and the resulting “butanologen NYLA84” was maintained onsynthetic complete media lacking histidine and uracil, and supplementedwith 1% ethanol at 30° C.

Expression Vector pLH468

The pLH468 plasmid (SEQ ID NO: 2) was constructed for expression ofDHAD, KivD, and HADH in yeast and is described in U.S. PatentApplication Publication No. 2009/0305363, herein incorporated byreference. pLH486 was constructed to contain: a chimeric gene having thecoding region of the ilvD gene from Streptococcus mutans (nt position3313-4849) expressed from the S. cerevisiae FBA1 promoter (nt 2109-3105)followed by the FBA1 terminator (nt 4858-5857) for expression of DHAD; achimeric gene having the coding region of codon optimized horse liveralcohol dehydrogenase (nt 6286-7413) expressed from the S. cerevisiaeGPM1 promoter (nt 7425-8181) followed by the ADH1 terminator (nt5962-6277) for expression of ADH; and a chimeric gene having the codingregion of the codon-optimized kivD gene from Lactococcus lactis (nt9249-10895) expressed from the TDH3 promoter (nt 10896-11918) followedby the TDH3 terminator (nt 8237-9235) for expression of KivD.

Coding regions for Lactococcus lactis ketoisovalerate decarboxylase(KivD) and horse liver alcohol dehydrogenase (HADH) were synthesized byDNA2.0, Inc. (Menlo Park, Calif.) based on codons that were optimizedfor expression in Saccharomyces cerevisiae (SEQ ID NO: 71 and 118,respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0.The encoded proteins are SEQ ID NOs: 117 and 119, respectively.Individual expression vectors for KivD and HADH were constructed. Toassemble pLH467 (pRS426::PTDH3-kivDy-TDH3t), vector pNY8 (SEQ ID NO:121; also named pRS426.GPD-ald-GPDt, described in U.S. PatentApplication Publication No. 2008/0182308, Example 17, which is hereinincorporated by reference) was digested with AscI and SfiI enzymes, thusexcising the GPD promoter and the ald coding region. A TDH3 promoterfragment (SEQ ID NO: 122) from pNY8 was PCR amplified to add an AscIsite at the 5′ end and an SpeI site at the 3′ end, using 5′ primerOT1068 and 3′ primer OT1067 (SEQ ID NOs: 123 and 124). The AscI/SfiIdigested pNY8 vector fragment was ligated with the TDH3 promoter PCRproduct digested with AscI and SpeI, and the SpeI-SfiI fragmentcontaining the codon optimized kivD coding region isolated from thevector pKivD-DNA2.0. The triple ligation generated vector pLH467(pRS426::PTDH3-kivDy-TDH3t). pLH467 was verified by restriction mappingand sequencing.

pLH435 (pRS425::PGPM1-Hadhy-ADH1t) was derived from vectorpRS425::GPM-sadB (SEQ ID NO: 78) which is described in U.S. ProvisionalApplication Ser. No. 61/058,970, Example 3, which is herein incorporatedby reference. pRS425::GPM-sadB is the pRS425 vector (ATCC No. 77106)with a chimeric gene containing the GPM1 promoter (SEQ ID NO:72), codingregion from a butanol dehydrogenase of Achromobacter xylosoxidans (sadB;DNA SEQ ID NO: 69; protein SEQ ID NO:70: disclosed in U.S. PatentApplication Publication No. 2009/0269823), and ADH1 terminator (SEQ IDNO: 73). pRS425::GPMp-sadB contains BbvI and PacI sites at the 5′ and 3′ends of the sadB coding region, respectively. A NheI site was added atthe 5′ end of the sadB coding region by site-directed mutagenesis usingprimers OT1074 and OT1075 (SEQ ID NOs: 126 and 127) to generate vectorpRS425-GPMp-sadB-NheI, which was verified by sequencing.pRS425::PGPM1-sadB-NheI was digested with NheI and PacI to drop out thesadB coding region, and ligated with the NheI-PacI fragment containingthe codon optimized HADH coding region from vector pHadhy-DNA2.0 tocreate pLH435.

To combine KivD and HADH expression cassettes in a single vector, yeastvector pRS411 (ATCC No. 87474) was digested with SacI and NotI, andligated with the SacI-SalI fragment from pLH467 that contains thePTDH3-kivDy-TDH3t cassette together with the SalI-NotI fragment frompLH435 that contains the PGPM1-Hadhy-ADH1t cassette in a triple ligationreaction. This yielded the vector pRS411::PTDH3-kivDy-PGPM1-Hadhy(pLH441) which was verified by restriction mapping.

In order to generate a co-expression vector for all three genes in thelower isobutanol pathway: ilvD, kivDy, and Hadhy, pRS423 FBA ilvD(Strep)(SEQ ID NO: 128) which is described in U.S. Patent ApplicationPublication No. 2010/0081154 as the source of the ilvD gene, was used.This shuttle vector contains an F1 origin of replication (nt 1423 to1879) for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426)for replication in yeast. The vector has an FBA1 promoter (nt 2111 to3108; SEQ ID NO: 120) and FBA terminator (nt 4861 to 5860; SEQ ID NO:129). In addition, it carries the His marker (nt 504 to 1163) forselection in yeast and ampicillin resistance marker (nt 7092 to 7949)for selection in E. coli. The ilvD coding region (nt 3116 to 4828; SEQID NO: 115; protein SEQ ID NO: 116) from Streptococcus mutans UA159(ATCC No. 700610) is between the FBA promoter and FBA terminator forminga chimeric gene for expression. In addition, there is a lumio tag fusedto the ilvD coding region (nt 4829-4849).

The first step was to linearize pRS423 FBA ilvD(Strep) (also calledpRS423-FBA(SpeI)-IlvD(Streptococcus mutans)-Lumio) with SacI and SacII(with SacII site blunt ended using T4 DNA polymerase), to give a vectorwith total length of 9,482 bp. The second step was to isolate thekivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI siteblunt ended using T4 DNA polymerase), which gives a 6,063 bp fragment.This fragment was ligated with the 9,482 bp vector fragment frompRS423-FBA(SpeI)-ilvD(Streptococcus mutans)-Lumio. This generated vectorpLH468 (pRS423::PFBA1-ilvD(Strep)Lumio-FBA1t-PTDH3-kivDy-TDH3t-PGPM1-hadhy-ADH1t) which was confirmed byrestriction mapping and sequencing.

pLH532 Construction

The pLH532 plasmid (SEQ ID NO: 130) was constructed for expression ofALS and KARI in yeast. pLH532 is a pHR81 vector (ATCC No. 87541)containing the following chimeric genes: 1) the CUP1 promoter (SEQ IDNO: 139), acetolactate synthase coding region from Bacillus subtilis(AlsS; SEQ ID NO: 137; protein SEQ ID NO: 138) and CYC1 terminator2 (SEQID NO: 133); 2) an ILV5 promoter (SEQ ID NO: 134), Pf5.1IvC codingregion (SEQ ID NO: 132) and ILV5 terminator (SEQ ID NO: 135); and 3) theFBA1 promoter (SEQ ID NO: 136), S. cerevisiae KARI coding region (ILV5;SEQ ID NO: 131); and CYC1 terminator.

The Pf5.IlvC coding region is a sequence encoding KARI derived fromPseudomonas fluorescens that was described in U.S. Patent ApplicationPublication No. 2009/0163376, which is herein incorporated by reference.

The Pf5.IlvC coding region was synthesized by DNA2.0, Inc. (Menlo Park,Calif.; SEQ ID NO: 132) based on codons that were optimized forexpression in Saccharomyces cerevisiae.

pYZ090 Construction

pYZ090 (SEQ ID NO: 1) is based on the pHR81 (ATCC No. 87541) backboneand was constructed to contain a chimeric gene having the coding regionof the alsS gene from Bacillus subtilis (nt position 457-2172) expressedfrom the yeast CUP1 promoter (nt 2-449) and followed by the CYC1terminator (nt 2181-2430) for expression of ALS, and a chimeric genehaving the coding region of the ilvC gene from Lactococcus lactis (nt3634-4656) expressed from the yeast ILV5 promoter (2433-3626) andfollowed by the ILV5 terminator (nt 4682-5304) for expression of KARI.

pYZ067 Construction

pYZ067 was constructed to contain the following chimeric genes: 1) thecoding region of the ilvD gene from S. mutans UA159 (nt position2260-3971) expressed from the yeast FBA1 promoter (nt 1161-2250)followed by the FBA terminator (nt 4005-4317) for expression ofdihydroxy acid dehydratase (DHAD), 2) the coding region for horse liverADH (nt 4680-5807) expressed from the yeast GPM promoter (nt 5819-6575)followed by the ADH1 terminator (nt 4356-4671) for expression of alcoholdehydrogenase, and 3) the coding region of the KivD gene fromLacrococcus lactis (nt 7175-8821) expressed from the yeast TDH3 promoter(nt 8830-9493) followed by the TDH3 terminator (nt 5682-7161) forexpression of ketoisovalerate decarboxylase.

pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadB and pLH475-Z4B8Construction

Construction of pRS423::CUP1-alsS+FBA-budA and pRS426::FBA-budC+GPM-sadBand pLH475-Z4B8 is described in U.S. Patent Application Publication No.2009/0305363, incorporated herein by reference.

Further, while various embodiments of the present invention have beendescribed above, it should be understood that they have been presentedby way of example only, and not limitation. It will be apparent topersons skilled in the relevant art that various changes in form anddetail can be made therein without departing from the spirit and scopeof the invention. Thus, the breadth and scope of the present inventionshould not be limited by any of the above-described exemplaryembodiments, but should be defined only in accordance with the claimsand their equivalents.

All publications, patents, and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patent,or patent application was specifically and individually indicated to beincorporated by reference.

EXAMPLES

The following nonlimiting examples will further illustrate theinvention. It should be understood that, while the following examplesinvolve corn as feedstock and COFA as carboxylic acid, other biomasssources can be used for feedstock and acids other than COFA can serve ascarboxylic acid, without departing from the present invention. Moreover,while the following examples involve butanol and butyl ester production,other alcohols including ethanol, and alcohol esters can be producedwithout departing from the present invention.

As used herein, the meaning of abbreviations used was as follows: “g”means gram(s), “kg” means kilogram(s), “L” means liter(s), “mL” meansmilliliter(s), “μL” means microliter(s), “mL/L” means milliliter(s) perliter, “mL/min” means milliliter(s) per min, “DI” means deionized, “uM”means micrometer(s), “nm” means nanometer(s), “w/v” means weight/volume,“OD” means optical density, “OD₆₀₀” means optical density at awavelength of 600 nM, “dcw” means dry cell weight, “rpm” meansrevolutions per minute, “° C.” means degree(s) Celsius, “° C./min” meansdegrees Celsius per minute, “slpm” means standard liter(s) per minute,“ppm” means part per million, “pdc” means pyruvate decarboxylase enzymefollowed by the enzyme number.

General Methods

Seed Flask Growth

A Saccharomyces cerevisiae strain that was engineered to produceisobutanol from a carbohydrate source, with pdc1 deleted, pdc5 deleted,and pdc6 deleted, was grown to 0.55-1.1 g/L dcw (OD₆₀₀ 1.3-2.6—ThermoHelios α Thermo Fisher Scientific Inc., Waltham, Mass.) in seed flasksfrom a frozen culture. The culture was grown at 26° C. in an incubatorrotating at 300 rpm. The frozen culture was previously stored at −80° C.The composition of the first seed flask medium was:

-   -   3.0 g/L dextrose    -   3.0 g/L ethanol, anhydrous    -   3.7 g/L ForMedium™ Synthetic Complete Amino Acid (Kaiser)        prop-Out: without HIS, without URA (Reference No. DSCK162CK)    -   6.7 g/L Difco Yeast Nitrogen Base without amino acids (No.        291920)

Twelve milliliters from the first seed flask culture was transferred toa 2 L flask and grown at 30° C. in an incubator rotating at 300 rpm. Thesecond seed flask has 220 mL of the following medium:

-   -   30.0 g/L dextrose    -   5.0 g/L ethanol, anhydrous    -   3.7 g/L ForMedium™ Synthetic Complete Amino Acid (Kaiser)        prop-Out: without HIS, without URA (Reference No. DSCK162CK)    -   6.7 g/L Difco Yeast Nitrogen Base without amino acids (No.        291920)    -   0.2 M MES Buffer titrated to pH 5.5-6.0

The culture was grown to 0.55-1.1 g/L dcw (OD₆₀₀ 1.3-2.6). An additionof 30 mL of a solution containing 200 g/L peptone and 100 g/L yeastextract was added at this cell concentration. Then, an addition of 300mL of 0.2 uM filter sterilized Cognis, 90-95% oleyl alcohol was added tothe flask. The culture continues to grow to >4 g/L dcw (OD₆₀₀>10) beforebeing harvested and added to the fermentation.

Fermentation Preparation

Initial Fermentation Vessel Preparation

A glass jacked, 2 L fermentation vessel (Sartorius AG, Goettingen,Germany) was charged with house water to 66% of the liquefaction weight.A pH probe (Hamilton Easyferm Plus K8, part number: 238627, HamiltonBonaduz AG, Bonaduz, Switzerland) was calibrated through the SartoriusDCU-3 Control Tower Calibration menu. The zero was calibrated at pH=7.The span was calibrated at pH=4. The probe was then placed into thefermentation vessel through the stainless steel head plate. A dissolvedoxygen probe (pO₂ probe) was also placed into the fermentation vesselthrough the head plate. Tubing used for delivering nutrients, seedculture, extracting solvent, and base were attached to the head plateand the ends were foiled. The entire fermentation vessel was placed intoa Steris (Steris Corporation, Mentor, Ohio) autoclave and sterilized ina liquid cycle for 30 minutes.

The fermentation vessel was removed from the autoclave and placed on aload cell. The jacket water supply and return line was connected to thehouse water and clean drain, respectively. The condenser cooling waterin and water out lines were connected to a 6-L recirculating temperaturebath running at 7° C. The vent line that transfers the gas from thefermentation vessel was connected to a transfer line that was connectedto a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc.,Waltham, Mass.). The sparger line was connected to the gas supply line.The tubing for adding nutrients, extract solvent, seed culture, and basewas plumbed through pumps or clamped closed.

The fermentation vessel temperature was controlled at 55° C. with athermocouple and house water circulation loop. Wet corn kernels (#2yellow dent) were ground using a hammer mill with a 1.0 mm screen, andthe resulting ground whole corn kernels were then added to thefermentation vessel at a charge that was 29-30% (dry corn solids weight)of the liquefaction reaction mass.

Lipase Treatment Pre-Liquefaction

A lipase enzyme stock solution was added to the fermentation vessel to afinal lipase concentration of 10 ppm. The fermentation vessel was heldat 55° C., 300 rpm, and 0.3 slpm N₂ overlay for >6 hrs. After the lipasetreatment was complete, liquefaction was performed as described below(Liquefaction).

Liquefaction

An alpha-amylase was added to the fermentation vessel per itsspecification sheet while the fermentation vessel was mixing at 300-1200rpm, with sterile, house N₂ being added at 0.3 slpm through the sparger.The temperature set-point was changed from 55° C. to 85° C. When thetemperature was >80° C., the liquefaction cook time was started and theliquefaction cycle was held at >80° C. for 90-120 minutes. Thefermentation vessel temperature set-point was set to the fermentationtemperature of 30° C. after the liquefaction cycle was complete. N₂ wasredirected from the sparger to the head space to prevent foaming withoutthe addition of a chemical antifoaming agent.

Lipase Treatment Post-Liquefaction

The fermentation vessel temperature was set to 55° C. instead of 30° C.after the liquefaction cycle was complete (Liquefaction). The pH wasmanually controlled at pH=5.8 by making bolus additions of acid or basewhen needed. A lipase enzyme stock solution was added to thefermentation vessel to a final lipase concentration of 10 ppm. Thefermentation vessel was held at 55° C., 300 rpm, and 0.3 slpm N₂ overlayfor >6 hrs. After the Lipase Treatment was complete, the fermentationvessel temperature was set to 30° C.

Lipase Heat Inactivation Treatment (Heat Kill Treatment Method)

The fermentation vessel temperature was held at >80° C. for >15 minutesto inactivate the lipase. After the Heat Inactivation Treatment wascomplete, the fermentation vessel temperature was set to 30° C.

Nutrient Addition Prior to Inoculation

Ethanol (6.36 mL/L, post-inoculation volume, 200 proof, anhydrous) wasadded to the fermentation vessel just prior to inoculation. Thiamine wasadded to a final concentration of 20 mg/L and 100 mg/L nicotinic acidwas also added just prior to inoculation.

Oleyl Alcohol or Corn Oil Fatty Acids Addition Prior to Inoculation

Added 1 L/L (post-inoculation volume) of oleyl alcohol or corn oil fattyacids immediately after inoculation.

Fermentation Vessel Inoculation

The fermentation vessels pO₂ probe was calibrated to zero while N₂ wasbeing added to the fermentation vessel. The fermentation vessels pO₂probe was calibrated to its span with sterile air sparging at 300 rpm.The fermentation vessel was inoculated after the second seed flaskwith >4 g/L dcw. The shake flask was removed from the incubator/shakerfor 5 minutes allowing a phase separation of the oleyl alcohol phase andthe aqueous phase. The aqueous phase (110 mL) was transferred to asterile, inoculation bottle. The inoculum was pumped into thefermentation vessel through a peristaltic pump.

Fermentation Vessel Operating Conditions

The fermentation vessel was operated at 30° C. for the entire growth andproduction stages. The pH was allowed to drop from a pH between 5.7-5.9to a control set-point of 5.2 without adding any acid. The pH wascontrolled for the remainder of the growth and production stage at apH=5.2 with ammonium hydroxide. Sterile air was added to thefermentation vessel, through the sparger, at 0.3 slpm for the remainderof the growth and production stages. The pO₂ was set to be controlled at3.0% by the Sartorius DCU-3 Control Box PID control loop, using stircontrol only, with the stirrer minimum being set to 300 rpm and themaximum being set to 2000 rpm. The glucose was supplied throughsimultaneous saccharification and fermentation of the liquified cornmash by adding a α-amylase (glucoamylase). The glucose was kept excess(1-50 g/L) for as long as starch was available for saccharification.

Analytical

Gas Analysis

Process air was analyzed on a Thermo Prima (Thermo Fisher ScientificInc., Waltham, Mass.) mass spectrometer. This was the same process airthat was sterilized and then added to each fermentation vessel. Eachfermentation vessel's off-gas was analyzed on the same massspectrometer. This Thermo Prima dB has a calibration check run everyMonday morning at 6:00 am. The calibration check was scheduled throughthe Gas Works v1.0 (Thermo Fisher Scientific Inc., Waltham, Mass.)software associated with the mass spectrometer. The gas calibrated forwere:

GAS Calibration Concentration mole % Cal Frequency Nitrogen 78% weeklyOxygen 21% weekly Isobutanol 0.2%  yearly Argon  1% weekly CarbonDioxide 0.03%   weekly

Carbon dioxide was checked at 5% and 15% during calibration cycle withother known bottled gases. Oxygen was checked at 15% with other knownbottled gases. Based on the analysis of the off-gas of each fermentationvessel, the amount of isobutanol stripped, oxygen consumed, and carbondioxide respired into the off-gas was measured by using the massspectrometer's mole fraction analysis and gas flow rates (mass flowcontroller) into the fermentation vessel. Calculate the gassing rate perhour and then integrating that rate over the course of the fermentation.

Biomass Measurement

A 0.08% Trypan Blue solution was prepared from a 1:5 dilution of 0.4%Trypan Blue in NaCl (VWR BDH8721-0) with 1×PBS. A 1.0 mL sample waspulled from a fermentation vessel and placed in a 1.5 mL Eppendorfcentrifuge tube and centrifuged in an Eppendorf, 5415C at 14,000 rpm for5 minutes. After centrifugation, the top solvent layer was removed withan m200 Variable Channel BioHit pipette with 20-200 μL BioHit pipettetips. Care was made not to remove the layer between the solvent andaqueous layers. Once the solvent layer was removed, the sample wasre-suspended using a Vortex-Genie® set at 2700 rpm.

A series of dilutions was required to prepare the ideal concentrationfor hemacytometer counts. If the OD was 10, a 1:20 dilution would beperformed to achieve 0.5 OD which would give the ideal amount of cellsto be counted per square, 20-30. In order to reduce inaccuracy in thedilution due to corn solids, multiple dilutions with cut 100-1000 μLBioHit pipette tips were required. Approximately, 1 cm was cut off thetips to increase the opening which prevented the tip from clogging. Fora 1:20 final dilution, an initial 1:1 dilution of fermentation sampleand 0.9% NaCl solution was prepared. Then, a 1:1 dilution of theprevious solution (i.e., the initial 1:1 dilution) and 0.9% NaClsolution (the second dilution) was generated followed by a 1:5 dilutionof the second dilution and Trypan Blue Solution. Samples were vortexedbetween each dilution and cut tips were rinsed into the 0.9% NaCl andTrypan Blue solutions.

The cover slip was carefully placed on top of the hemacytometer (HausserScientific Bright-Line 1492). An aliquot (10 μL) was drawn of the finalTrypan Blue dilution with an m20 Variable Channel BioHit pipette with2-20 μL BioHit pipette tips and injected into the hemacytometer. Thehemacytometer was placed on the Zeis Axioskop 40 microscope at 40×magnification. The center quadrant was broken into 25 squares and thefour corner and center squares in both chambers were then counted andrecorded. After both chambers were counted, the average was taken andmultiplied by the dilution factor (20), then by 25 for the number forsquares in the quadrant in the hemacytometer, and then divided by 0.0001mL which is the volume of the quadrant that was counted. The sum of thiscalculation is the number cells per mL.

LC Analysis of Fermentation Products in the Aqueous Phase

Samples were refrigerated until ready for processing. Samples wereremoved from refrigeration and allowed to reach room temperature (aboutone hour). Approximately 300 μL of sample was transferred with a m1000Variable Channel BioHit pipette with 100-1000 μL BioHit pipette tipsinto a 0.2 um centrifuge filter (Nanosep® MF modified nylon centrifugefilter), then centrifuged using a Eppendorf, 5415C for five minutes at14,000 rpm. Approximately 200 μL of filtered sample was transferred intoa 1.8 auto sampler vial with a 250 μL glass vial insert with polymerfeet. A screw cap with PTFE septa was used to cap the vial beforevortexing the sample with a Vortex-Genie® set at 2700 rpm.

Sample was then run on Agilent 1200 series LC equipped with binary,isocratic pumps, vacuum degasser, heated column compartment, samplercooling system, UV DAD detector and RI detector. The column used was anAminex HPX-87H, 300×7.8 with a Bio-Rad Cation H refill, 30×4.6 guardcolumn. Column temperature was 40° C., with a mobile phase of 0.01 Nsulfuric acid, at a flow rate of 0.6 mL/min for 40 minutes. Results areshown in Table 1.

TABLE 1 Retention times of fermentation products in aqueous phase UVHPLC 302/310 RID Range of Retention Normalized to 10 μL RetentionStandards, Time, injections FW Time, min g/L min citric acid 192.128.025 0.3-17 7.616 glucose 180.16 8.83 0.5-71 pyruvic acid (Na) 110.049.388  0.1-5.2 8.5 A-Kiv (Na) 138.1 9.91 0.07-5.0  8.552,3-dihydroxyisovaleric 156.1 10.972  0.2-8.8 10.529 acid (Na) succinicacid 118.09 11.561 0.3-16 11.216 lactic acid (Li) 96.01 12.343 0.3-1711.948 glycerol 92.09 12.974 0.8-39 formic acid 46.03 13.686 0.2-1313.232 acetate (Na) 82.03 14.914 0.5-16 14.563 meso-butanediol 90.1217.583 0.1-19 (+/−)-2,3-butanediol 90.12 18.4 0.2-19 isobutyric acid88.11 19.685  0.1-8.0 19.277 ethanol 46.07 21.401 0.5-34isobutyraldehyde 72.11 27.64  0.01-0.11 isobutanol 74.12 32.276 0.2-153-OH-2-butanone (acetoin) 88.11 0.1-11 17.151GC Analysis of Fermentation Products in the Solvent Phase

Samples were refrigerated until ready for processing. Samples wereremoved from refrigeration and allowed to reach room temperature (aboutone hour). Approximately 150 μL of sample was transferred using a m1000Variable Channel BioHit pipette with 100-1000 μL BioHit pipette tipsinto a 1.8 auto sampler vial with a 250 μL glass vial insert withpolymer feet. A screw cap with PTFE septa was used to cap the vial.

Sample was then run on Agilent 7890A GC with a 7683B injector and aG2614A auto sampler. The column was a HP-InnoWax column (30 m×0.32 mmID, 0.25 μm film). The carrier gas was helium at a flow rate of 1.5mL/min measured at 45° C. with constant head pressure; injector splitwas 1:50 at 225° C.; oven temperature was 45° C. for 1.5 minutes, 45° C.to 160° C. at 10° C./min for 0 minutes, then 230° C. at 35° C./min for14 minutes for a run time of 29 minutes. Flame ionization detection wasused at 260° C. with 40 mL/min helium makeup gas. Results are shown inTable 2.

TABLE 2 Retention times of fermentation products in solvent phase. GC302/310 Solvent Normalized to 10 μL Retention Range of Standards,injections FW Time, min g/L isobutyraldehyde 72.11 2.75   0.7-10.4ethanol 46.07 3.62 0.5-34 isobutanol 74.12 5.53 0.2-16 3-OH-2-butanone(acetoin) 88.11 8.29 0.1-11 (+/−)-2,3-butanediol 90.12 10.94 0.1-19isobutyric acid 88.11 11.907  0.1-7.9 meso-butanediol 90.12 11.26 0.1-6.5 glycerol 92.09 16.99 0.8-9 

Samples analyzed for fatty acid butyl esters were run on Agilent 6890 GCwith a 7683B injector and a G2614A auto sampler. The column was aHP-DB-FFAP column (15 meters×0.53 mm ID (Megabore), 1-micron filmthickness column (30 m×0.32 mm ID, 0.25 μm film). The carrier gas washelium at a flow rate of 3.7 mL/min measured at 45° C. with constanthead pressure; injector split was 1:50 at 225° C.; oven temperature was100° C. for 2.0 minutes, 100° C. to 250° C. at 10° C./min, then 250° C.for 9 minutes for a run time of 26 minutes. Flame ionization detectionwas used at 300° C. with 40 mL/min helium makeup gas. The following GCstandards (Nu-Chek Prep; Elysian, Minn.) were used to confirm theidentity of fatty acid isobutyl ester products: iso-butyl palmitate,iso-butyl stearate, iso-butyl oleate, iso-butyl linoleate, iso-butyllinolenate, iso-butyl arachidate.

Examples 1-14 describe various fermentation conditions that may be usedfor the claimed methods. As an example, some fermentations weresubjected to Lipase Treatment pre-liquefaction and others were subjectedto Lipase Treatment post-liquefaction. In other examples, thefermentation was subjected to Heat inactivation Treatment. Followingfermentation, the effective isobutanol titer (Eff Iso Titer) wasmeasured, that is, the total grams of isobutanol produced per literaqueous volume. Results are shown in Table 3.

Example 1 Control

Experiment identifier 2010Y014 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Nutrient Addition Prior to Inoculation method, Fermentation VesselInoculation method, Fermentation Vessel Operating Conditions method, andall of the Analytical methods. Oleyl alcohol was added in a single batchbetween 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.

Example 2

Experiment identifier 2010Y015 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Lipase Treatment Post-Liquefaction method, Nutrient Addition Prior toInoculation method, Fermentation Vessel Inoculation method, FermentationVessel Operating Conditions method, and all of the Analytical methods.Oleyl alcohol was added in a single batch between 0.1-1.0 hr afterinoculation. The butanologen was NGCI-070.

Example 3

Experiment identifier 2010Y016 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Lipase Treatment Post-Liquefaction method, Nutrient Addition Prior toInoculation method with the exception of the exclusion of ethanol,Fermentation Vessel Inoculation method, Fermentation Vessel OperatingConditions method, and all of the Analytical methods. Oleyl alcohol wasadded in a single batch between 0.1-1.0 hr after inoculation. Thebutanologen was NGCI-070.

Example 4

Experiment identifier 2010Y017 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Heat Kill Treatment method Post-Liquefaction, Nutrient Addition Prior toInoculation method with the exception of the exclusion of ethanol,Fermentation Vessel Inoculation method, Fermentation Vessel OperatingConditions method, and all of the Analytical methods. Oleyl alcohol wasadded in a single batch between 0.1-1.0 hr after inoculation. Thebutanologen was NGCI-070.

Example 5

Experiment identifier 2010Y018 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Lipase Treatment Post-Liquefaction method with the exception of onlyadding 7.2 ppm lipase after liquefaction, Heat Kill Treatment methodPost-Liquefaction, Nutrient Addition Prior to Inoculation method,Fermentation Vessel Inoculation method, Fermentation Vessel OperatingConditions method, and all of the Analytical methods. Oleyl alcohol wasadded in a single batch between 0.1-1.0 hr after inoculation. Thebutanologen was NGCI-070.

Example 6 Control

Experiment identifier 2010Y019 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Heat Kill Treatment method Post-Liquefaction, Nutrient Addition Prior toInoculation method, Fermentation Vessel Inoculation method, FermentationVessel Operating Conditions method, and all of the Analytical methods.Oleyl alcohol was added in a single batch between 0.1-1.0 hr afterinoculation. The butanologen was NGCI-070.

Example 7 Control

Experiment identifier 2010Y021 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Lipase TreatmentPre-Liquefaction method, Liquefaction method, Heat Kill Treatment duringliquefaction, Nutrient Addition Prior to Inoculation method,Fermentation Vessel Inoculation method, Fermentation Vessel OperatingConditions method, and all of the Analytical methods. Oleyl alcohol wasadded in a single batch between 0.1-1.0 hr after inoculation. Thebutanologen was NGCI-070.

Example 8

Experiment identifier 2010Y022 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Nutrient Addition Prior to Inoculation method, Fermentation VesselInoculation method, Fermentation Vessel Operating Conditions method, andall of the Analytical methods. Oleyl alcohol was added in a single batchbetween 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.

Example 9

Experiment identifier 2010Y023 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Lipase Treatment Post-Liquefaction method, no Heat Kill Treatment,Nutrient Addition Prior to Inoculation method, Fermentation VesselInoculation method, Fermentation Vessel Operating Conditions method, andall of the Analytical methods. Corn oil fatty acids made from crude cornoil was added in a single batch between 0.1-1.0 hr after inoculation.The butanologen was NGCI-070.

Example 10

Experiment identifier 2010Y024 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Lipase TreatmentPre-Liquefaction method, Liquefaction method, Heat Kill Treatment duringliquefaction, Nutrient Addition Prior to Inoculation method with theexception of there being no addition of ethanol, Fermentation VesselInoculation method, Fermentation Vessel Operating Conditions method, andall of the Analytical methods. Oleyl alcohol was added in a single batchbetween 0.1-1.0 hr after inoculation. The butanologen was NGCI-070.

Example 11

Experiment identifier 2010Y029 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Lipase TreatmentPre-Liquefaction method, Liquefaction method, Heat Kill Treatment duringliquefaction, Nutrient Addition Prior to Inoculation method,Fermentation Vessel Inoculation method, Fermentation Vessel OperatingConditions method, and all of the Analytical methods. Corn oil fattyacids made from crude corn oil was added in a single batch between0.1-1.0 hr after inoculation. The butanologen was NGCI-070.

Example 12

Experiment identifier 2010Y030 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Lipase TreatmentPre-Liquefaction method, Liquefaction method, Heat Kill Treatment duringliquefaction, Nutrient Addition Prior to Inoculation method with theexception of there being no addition of ethanol, Fermentation VesselInoculation method, Fermentation Vessel Operating Conditions method, andall of the Analytical methods. Corn oil fatty acids made from crude cornoil was added in a single batch between 0.1-1.0 hr after inoculation.The butanologen was NGCI-070.

Example 13 Control

Experiment identifier 2010Y031 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Lipase Treatment Post Liquefaction method, no Heat Kill Treatment,Nutrient Addition Prior to Inoculation method with the exception ofthere being no addition of ethanol, Fermentation Vessel Inoculationmethod, Fermentation Vessel Operating Conditions method, and all of theAnalytical methods. Corn oil fatty acids made from crude corn oil wasadded in a single batch between 0.1-1.0 hr after inoculation. Thebutanologen was NGCI-070.

Example 14

Experiment identifier 2010Y032 included: Seed Flask Growth method,Initial Fermentation Vessel Preparation method, Liquefaction method,Lipase Treatment Post-Liquefaction method, no Heat Kill Treatment,Nutrient Addition Prior to Inoculation method, Fermentation VesselInoculation method, Fermentation Vessel Operating Conditions method, andall of the Analytical methods. Corn oil fatty acids made from crude cornoil was added in a single batch between 0.1-1.0 hr after inoculation.The butanologen was NGCI-070.

TABLE 3 Fermentation conditions for Examples 1-14. Max cell Eff Iso maxEff Experimental Count × Ethanol Heat Kill Titer Iso rate Example #Identifier Lipase 10⁷ g/L Solvent Lipase g/L* g/L/h 1 2010Y014 none 27.25 Oleyl none 56.0 0.79 alcohol 2 2010Y015 10 ppm 31.5 5 Oleyl none 52.40.74 alcohol 3 2010Y016 10 ppm 6.7 0 Oleyl none 25.9 0.36 alcohol 42010Y017 none 7.9 0 Oleyl post- 17.2 0.25 alcohol liquefaction 52010Y018 7.2 ppm  16.2 5 Oleyl post- 45.8 0.66 alcohol liquefaction 62010Y019 none 17.5 5 Oleyl post- 48.1 0.69 alcohol liquefaction 72010Y021 10 ppm 21.2 5 Oleyl during 46.8 0.82 alcohol liquefaction 82010Y022 none 9 5 Oleyl during 56.2 0.87 alcohol liquefaction 9 2010Y02310 ppm 12.8 5 Corn Oil none 60.3 1.3 Fatty Acids 10 2010Y024 10 ppm 25.30 Oleyl during 19.8 0.33 alcohol liquefaction 11 2010Y029 10 ppm 21.2 5Corn Oil during 28.36 0.52 Fatty liquefaction Acids 12 2010Y030 10 ppm 90 Corn Oil during 12.71 0.24 Fatty liquefaction Acids 13 2010Y031 10 ppm12.8 0 Corn Oil none 18.86 0.35 Fatty Acids 14 2010Y032 10 ppm 25.3 5Corn Oil none 53.36 0.92 Fatty Acids *The “Eff Iso Titer g/L” = totalgrams of isobutanol produced per liter aqueous volume

Example 15

The experimental identifier was GLNOR432A. NYLA74 (a butanediolproducer—NGCI-047) was grown in 25 mL of medium in a 250 mL flask from afrozen vial to ˜10D. The pre-seed culture was transferred to a 2 L flaskand grown to 1.7-1.8 OD. The medium for both flasks was:

3.0 g/L dextrose

3.0 g/L ethanol, anhydrous

6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)

1.4 g/L Yeast propout Mix (Sigma Y2001)

10 mL/L1% w/v L-Leucine stock solution

2 mL/L1% w/v L-Tryptophan stock solution

A 1 L, Applikon fermentation vessel was inoculated with 60 mL of theseed flask. The fermentation vessel contained 700 mL of the followingsterile medium:

20.0 g/L dextrose

8.0 mL/L ethanol, anhydrous

6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920)

2.8 g/L Yeast propout Mix (Sigma Y2001)

20 mL/L 1% w/v L-Leucine stock solution

4 mL/L 1% w/v L-Tryptophan stock solution

0.5 mL Sigma 204 Antifoam

0.8 mL/L 1% w/v Ergesterol solution in 1:1::Tween 80:Ethanol

The residual glucose was kept excess with a 50% w/w glucose solution.The dissolved oxygen concentration of the fermentation vessel wascontrolled at 30% with stir control. The pH was controlled at pH=5.5.The fermentation vessel was sparged with 0.3 slpm of sterile, house air.The temperature was controlled at 30° C.

Example 16

The experimental identifier was GLNOR434A. This example is the same asexample 15 with the exception of the addition of 3 g of oleic acid andthe addition of 3 g of palmitic acid prior to inoculation. NYLA74 (abutanediol producer—NGCI-047) was the biocatalyst.

FIG. 7 shows that there were more grams per liter of glucose consumed inthe fermentation vessel that received the fatty acids. The squaresrepresent the fermentation vessel that received oleic acid and palmiticacid. The circles represent the fermentation vessel that did not receiveany extra fatty acids.

Example 17

The experimental identifier was GLNOR435A. This example was the same asexample 15 except it was inoculated with NYLA74 (an isobutanolproducer—NGCI-049).

Example 18

The experimental identifier was GLNOR437A. This example was the same asExample 16 except it was inoculated with NYLA74 (an isobutanol producer)(NGCI-049).

FIG. 8 shows that there were more grams per liter of glucose consumed inthe fermentation vessel that received the fatty acids. The squaresrepresent the fermentation vessel that received oleic acid and palmiticacid. The circles represent the fermentation vessel that did not receiveany extra fatty acids.

Example 19

The experimental identifier was 090420_(—)3212. This example was runsimilarly to Example 15 except it was inoculated with butanologen NYLA84(an isobutanol producer). This fermentation was run in a 1 L Sartoriusfermentation vessel.

Example 20

The experimental identifier was 2009Y047. This example was run similarlyto Example 16 except it was inoculated with butanologen NYLA84 (anisobutanol producer). This fermentation was run in a 1 L Sartoriusfermentation.

FIG. 9 shows that there were more grams per liter of glucose consumed inthe fermentation vessel that received the fatty acids. The squaresrepresent the fermentation vessel that received oleic acid and palmiticacid. The circles represent the fermentation vessel that did not receiveany extra fatty acids.

Table 4 shows +/− fatty acid addition, maximum optical density, and g/Lglucose consumed.

TABLE 4 Fatty 69 hours g/L Experimental Acids 69 hours glucose Example #Identifier Strain Added Product OD₆₀₀ consumed 15 GLNOR432A NYLA74 −butanediol 12.8 86.0 16 GLNOR434A NYLA74 + butanediol 23.1 95.9 17GLNOR435A NYLA74 − isobutanol 2.4 16.9 18 GLNOR437A NYLA74 + isobutanol4.5 18.3 19 090420_3212 NYLA84 − isobutanol 9.6 39.3 20 2009Y047NYLA84 + isobutanol 20.2 49.1

Example 21 Lipase Treatment of Liquefied Corn Mash for SimultaneousSaccharification and Fermentation with In-Situ Product Removal UsingOleyl Alcohol

Samples of broth and oleyl alcohol taken from fermentations run asdescribed above in Examples 1, 2, and 3 were analyzed for wt % lipid(derivatized as fatty acid methyl esters, FAME) and for wt % free fattyacid (FFA, derivatized as fatty acid methyl esters, FAME) according tothe method described by E. G. Bligh and W. J. Dyer (Canadian Journal ofBiochemistry and Physiology, 37:911-17, 1959, hereafter Reference 1).The liquefied corn mash that was prepared for each of the threefermentations was also analyzed for wt % lipid and for wt % FFA aftertreatment with Lipolase® 100 L (Novozymes) (10 ppm of Lipolase® totalsoluble protein (BCA protein analysis, Sigma Aldrich)) per kg ofliquefaction reaction mass containing 30 wt % ground corn kernels). Nolipase was added to the liquefied corn mash in Example 1 (control), andthe fermentations described in Examples 2 and 3 containing liquefiedcorn mash treated with lipase (no heat inactivation of lipase) wereidentical except that no ethanol was added to the fermentation describedin Example 3.

The % FFA in lipase-treated liquefied corn mash prepared forfermentations run as described in Examples 2 and 3 was 88% and 89%,respectively, compared to 31% without lipase treatment (Example 1). At70 h (end of run (EOR)), the concentration of FFA in the OA phase offermentations run as described in Examples 2 and 3 (containing activelipase) was 14% and 20%, respectively, and the corresponding increase inlipids (measured as corn oil fatty acid methyl ester derivatives) wasdetermined by GC/MS to be due to the lipase-catalyzed esterification ofCOFA by OA, where COFA was first produced by lipase-catalyzed hydrolysisof corn oil in the liquefied corn mash. Results are shown in Table 5.

TABLE 5 Lipid and free fatty acid content of fermentations containingoleyl alcohol as ISPR solvent and active lipase time (h), lipids FFAlipids FFA lipids + fermentation lipase sample (wt %) (wt %) (g) (g) FFA(g) % FFA Example 1 none liq. mash 0.61 0.28 5.3 2.4 7.7 31 Example 1none 0.8 h, broth 0.49 0.22 5.5 2.5 8.0 31 Example 1 none  31 h, broth0.19 0.03 2.1 0.3 2.4 13 Example 1 none  31 h, OA 0.36 0.21 3.4 2.0 5.337 Example 1 none  70 h, broth 0.15 0.03 1.7 0.3 2.0 15 Example 1 none 70 h, OA 0.57 0.25 5.3 2.3 7.7 31 Example 2 10 ppm liq. mash 0.13 0.971.1 8.5 9.6 88 Example 2 10 ppm 0.8 h, broth 0.15 0.62 1.7 7.0 8.7 81Example 2 10 ppm  31 h, broth 0.16 0.05 1.8 0.5 2.3 23 Example 2 10 ppm 31 h, OA 0.37 0.23 3.5 2.2 5.7 38 Example 2 10 ppm  70 h, broth 0.170.02 1.9 0.3 2.2 13 Example 2 10 ppm  70 h, OA 0.60 0.10 5.7 1.0 6.7 14Example 3 10 ppm liq. mash 0.12 0.97 1.0 8.5 9.5 89 Example 3 10 ppm 0.8h, broth 0.32 0.40 3.6 4.5 8.1 56 Example 3 10 ppm  31 h, broth 0.170.05 1.9 0.6 2.5 24 Example 3 10 ppm  31 h, OA 0.38 0.22 3.6 2.1 5.7 37Example 3 10 ppm  70 h, broth 0.15 0.02 1.7 0.2 1.9 13 Example 3 10 ppm 70 h, OA 0.46 0.12 4.4 1.1 5.6 20

Example 22 Heat Inactivation of Lipase in Lipase-Treated Liquefied CornMash to Limit Production of Oleyl Alcohol Esters of Corn Oil Free FattyAcids

Tap water (918.4 g) was added to a jacketed 2-L resin kettle, then 474.6g wet weight (417.6 g dry weight) of ground whole corn kernels (1.0 mmscreen on hammer mill) was added with stirring. The mixture was heatedto 55° C. with stirring at 300 rpm, and the pH adjusted to 5.8 with 2 Nsulfuric acid. To the mixture was added 14.0 g of an aqueous solutioncontaining 0.672 g of Spezyme®-FRED L (Genencor®, Palo Alto, Calif.),and the temperature of the mixture increased to 85° C. with stirring at600 rpm and pH 5.8. After 120 minutes at 85° C., the mixture was cooledto 50° C. and 45.0 mL aliquots of the resulting liquefied corn mash weretransferred to 50-mL polypropylene centrifuge tubes and stored frozen at−80° C.

In a first reaction, 50 g of liquefied corn mash prepared as describedabove was mixed with 10 ppm Lipolase® 100 L (Novozymes) for 6 h at 55°C. and with no inactivation of lipase at 85° C. for 1 h, the mixture wascooled to 30° C. In a second reaction, 50 g of liquefied corn mash wasmixed with 10 ppm Lipolase® for 6 h at 55° C., then heated to 85° C. for1 h (lipase inactivation), then cooled to 30° C. In a third reaction, 50g of liquefied corn mash without added lipase was mixed for 6 h at 55°C., and with no heating at 85° C. for 1 h, the mixture was cooled to 30°C., 38 g of oleyl alcohol was added, and the resulting mixture stirredfor 73 h at 30° C. In a fourth reaction, 50 g of liquefied corn mashwithout added lipase was mixed for 6 h at 55° C., then heated to 85° C.for 1 h, then cooled to 30° C. Each of the four reaction mixtures wassampled at 6 h, then 38 g of oleyl alcohol added, and the resultingmixtures stirred at 30° C. and sampled at 25 h and 73 h. Samples (bothliquefied mash and oleyl alcohol (OA)) were analyzed for wt % lipid(derivatized as fatty acid methyl esters, FAME) and for wt % free fattyacid (FFA, derivatized as fatty acid methyl esters, FAME) according tothe method described by Reference 1.

The % FFA in the OA phase of the second reaction run with heatinactivation of lipase prior to OA addition was 99% at 25 h and 95% at73 h, compared to only 40% FFA and 21% FFA at 25 h and 73 h,respectively, when the lipase in lipase-treated liquefied corn mash wasnot heat inactivated (first reaction). No significant change in % FFAwas observed in the two control reactions without added lipase. Resultsare shown in Table 6.

TABLE 6 Lipid and free fatty acid content of a mixture of liquefied cornmash and oleyl alcohol in the presence or absence of active orheat-inactivated lipase reaction time (h), lipids FFA lipids FFA lipid +FFA conditions sample (wt %) (wt %) (mg) (mg) (mg) % FFA 10 ppm activelipase,  6 h, liq. mash 0.08 0.71 41 345 386 89 no 85° C. heat treatment25 h, liq. mash 0.22 0.06 105 27 132 20 25 h, OA 0.58 0.39 212 143 35540 73 h, liq. mash 0.25 0.05 121 22 143 18 73 h, OA 0.91 0.24 333 88 42021 10 ppm inactive lipase,  6 h, lip. mash 0.06 0.45 28 224 252 89 85°C. heat treatment 25 h, lip. mash 0.10 0.11 49 54 103 53 25 h, OA 0.020.96 8 366 374 99 73 h, lip. mash 0.24 0.15 117 72 189 62 73 h, OA 0.061.11 23 424 447 95 no lipase,  6 h, lip. mash 0.80 0.40 401 199 599 33no 85° C. heat treatment 25 h, lip. mash 0.30 0.05 147 25 173 15 25 h,OA 0.55 0.36 212 139 351 40 73 h, lip. mash 0.23 0.05 117 26 143 23 73h, OA 0.79 0.42 305 162 467 34 no lipase,  6 h, lip. mash 0.74 0.36 370183 553 33 85° C. heat treatment 25 h, lip. mash 0.31 0.05 156 27 183 1525 h, OA 0.60 0.35 233 136 369 37 73 h, lip. mash 0.20 0.05 99 23 121 2373 h, OA 0.84 0.41 326 159 486 33

Example 23 Heat Inactivation of Lipase in Lipase-Treated Liquefied CornMash for Simultaneous Saccharification and Fermentation with In-SituProduct Removal Using Oleyl Alcohol

Three fermentations were run as described above in Examples 4, 5, and 6.No lipase was added to the liquefied corn mash in Examples 4 and 6 priorto fermentation, and the Lipase Treatment of the liquefied corn mash inthe fermentation described in Example 5 (using 7.2 ppm of Lipolase®total soluble protein) was followed immediately by Heat InactivationTreatment (to completely inactivate the lipase), and subsequentlyfollowed by Nutrient Addition Prior to Inoculation and fermentation. The% FFA in liquefied corn mash prepared without lipase treatment forfermentations run as described in Examples 4 and 6 was 31% and 34%,respectively, compared to 89% with lipase treatment (Example 5). Overthe course of the fermentations listed in Table 10, the concentration ofFFA in the OA phase did not decrease in any of the three fermentations,including that containing heat-inactivated lipase. The % FFA in the OAphase of the fermentation run according to Example 5 (with heatinactivation of lipase prior to fermentation) was 95% at 70 h (end ofrun (EOR)), compared to only 33% FFA for the remaining two fermentations(Examples 4 and 6) where liquefied corn mash was not treated withlipase. Results are shown in Table 7.

TABLE 7 Lipid and free fatty acid content of fermentations containingoleyl alcohol as ISPR solvent and heat-inactivated lipase (after lipasetreatment of liquefied mash) time (h), lipids FFA lipids FFA lipid +fermentation lipase sample (wt %) (wt %) (g) (g) FFA (g) % FFA Example 4none liquefied mash 0.65 0.30 7.2 3.3 10.4 31 Example 4 none 0.2 h,broth 0.56 0.28 6.6 3.3 9.9 33 Example 4 none 4.3 h, broth 0.28 0.09 3.31.0 4.4 24 Example 4 none 4.3 h, OA 0.45 0.27 4.0 2.4 6.4 37 Example 4none  30 h, broth 0.17 0.05 2.0 0.6 2.7 24 Example 4 none  30 h, OA 0.630.29 5.7 2.6 8.3 32 Example 4 none  53 h, broth 0.13 0.04 1.5 0.5 2.0 23Example 4 none  53 h, OA 0.67 0.32 6.0 2.9 8.9 32 Example 4 none  70 h,broth 0.13 0.04 1.5 0.4 1.9 23 Example 4 none  70 h, OA 0.64 0.31 5.82.8 8.5 33 Example 5 7.2 ppm liquefied mash 0.11 0.89 1.3 9.9 11.2 89Example 5 7.2 ppm 0.2 h, broth 0.25 0.83 2.9 9.8 12.8 77 Example 5 7.2ppm 4.3 h, broth 0.14 0.17 1.6 2.1 3.7 56 Example 5 7.2 ppm 4.3 h, OA0.02 0.84 0.2 7.9 8.1 97 Example 5 7.2 ppm  30 h, broth 0.08 0.18 1.02.1 3.1 68 Example 5 7.2 ppm  30 h, OA 0.04 0.92 0.3 8.6 8.9 96 Example5 7.2 ppm  53 h, broth 0.07 0.11 0.9 1.3 2.2 61 Example 5 7.2 ppm  53 h,OA 0.08 0.95 0.7 8.9 9.6 93 Example 5 7.2 ppm  70 h, broth 0.08 0.10 0.91.2 2.1 55 Example 5 7.2 ppm  70 h, OA 0.05 0.94 0.4 8.8 9.2 95 Example6 none liquefied mash 0.66 0.34 7.3 3.8 11.1 34 Example 6 none 0.2 h,broth 0.63 0.34 7.6 4.0 11.6 34 Example 6 none 4.3 h, broth 0.33 0.103.9 1.2 5.1 23 Example 6 none 4.3 h, OA 0.45 0.27 4.0 2.4 6.4 38 Example6 none  30 h, broth 0.17 0.06 2.1 0.8 2.8 26 Example 6 none  30 h, OA0.69 0.33 6.2 3.0 9.1 32 Example 6 none  53 h, broth 0.14 0.05 1.6 0.52.2 25 Example 6 none  53 h, OA 0.72 0.35 6.4 3.1 9.5 33 Example 6 none 70 h, broth 0.15 0.05 1.8 0.6 2.4 25 Example 6 none  70 h, OA 0.70 0.346.2 3.0 9.2 33

Example 24 Lipase Treatment of Ground Whole Corn Kernels Prior toLiquefaction

Tap water (1377.6 g) was added into each of two jacketed 2-L resinkettles, then 711.9 g wet weight (625.8 g dry weight) of ground wholecorn kernels (1.0 mm screen on hammer mill) was added to each kettlewith stirring. Each mixture was heated to 55° C. with stirring at 300rpm, and the pH adjusted to 5.8 with 2 N sulfuric acid. To each mixturewas added 21.0 g of an aqueous solution containing 1.008 g ofSpezyme®-FRED L (Genencor®, Palo Alto, Calif.). To one mixture was thenadded 10.5 mL of aqueous solution of Lipolase® 100L Solution (21 mgtotal soluble protein, 10 ppm lipase final concentration) and to thesecond mixture was added 1.05 mL of aqueous solution of Lipolase® 100LSolution (2.1 mg total soluble protein, 1.0 ppm lipase finalconcentration). Samples were withdrawn from each reaction mixture at 1h, 2 h, 4 h and 6 h at 55° C., then the temperature of the mixture wasincreased to 85° C. with stirring at 600 rpm and pH 5.8, and a samplewas taken when the mixture first reached 85° C. After 120 minutes at 85°C., a sample was taken and the mixtures were cooled to 50° C. and finalsamples of the resulting liquefied corn mash were transferred to 50-mLpolypropylene centrifuge tubes; all samples were stored frozen at −80°C.

In two separate reactions, a 50 g sample of the 10 ppm lipase-treatedliquefied corn mash or a 55 g sample of the 1.0 ppm lipase-treatedliquefied corn mash prepared as described above was mixed with oleylalcohol (OA) (38 g) at 30° C. for 20 h, then the liquefied mash and OAin each reaction mixture were separated by centrifugation and each phaseanalyzed for wt % lipid (derivatized as fatty acid methyl esters, FAME)and for wt % free fatty acid (FFA, derivatized as fatty acid methylesters, FAME) according to the method described by Reference 1. The %FFA in the OA phase of the liquefied mash/OA mixture prepared using heatinactivation of 10 ppm lipase during liquefaction was 98% at 20 h,compared to only 62% FFA in the OA phase of the liquefied mash/OAmixture prepared using heat inactivation of 1.0 ppm lipase duringliquefaction. Results are shown in Table 8.

TABLE 8 Lipid and free fatty acid content of a mixture of liquefied cornmash and oleyl alcohol, using lipase treatment of ground corn suspensionprior to liquefaction (heat inactivation of lipase during liquefaction)reaction lipids FFA lipids FFA lipid + FFA conditions time (h), sample(wt %) (wt %) (mg) (mg) (mg) % FFA 10 ppm lipase  1 h, pre-liquefaction0.226 0.627 112 311 424 74 at 55° C. prior to  2 h, pre-liquefaction0.199 0.650 99 323 422 77 liquefaction at  4 h, pre-liquefaction 0.1510.673 75 334 410 82 85° C., mix with  6 h, pre-liquefaction 0.101 0.70050 348 398 87 OA for 20 h  0 h, 85° C., liq. mash 0.129 0.764 64 380 44486  2 h, 85° C., liq. mash 0.129 0.751 64 373 437 85 20 h, 30° C., liq.mash 0.074 0.068 37 34 71 48 20 h, 30° C., OA 0.015 1.035 5.7 394 400 981.0 ppm lipase  1 h, pre-liquefaction 0.408 0.480 226 266 492 54 at 55°C. prior to  2 h, pre-liquefaction 0.401 0.424 222 235 457 51liquefaction at  4 h, pre-liquefaction 0.299 0.433 165 240 405 58 85°C., mix with  6 h, pre-liquefaction 0.346 0.453 192 251 442 57 OA for 20h  0 h, 85° C., liq. mash 0.421 0.407 233 225 458 49  2 h, 85° C., liq.mash 0.424 0.429 235 237 472 50 20 h, 30° C., liq. mash 0.219 0.054 12130 151 20 20 h, 30° C., OA 0.344 0.573 140 233 373 62

Example 25 Lipase Screening for Treatment of Ground Whole Corn KernelsPrior to Liquefaction

Seven reaction mixtures containing tap water (67.9 g) and ground wholecorn kernels (35.1 g wet wt., ground with 1.0 mm screen using a hammermill) at pH 5.8 were stirred at 55° C. in stoppered flasks. A 3-mLsample (t=0 h) was removed from each flask and the sample immediatelyfrozen on dry ice, then ca. 0.5 mL of 10 mM sodium phosphate buffer (pH7.0) containing 1 mg total soluble protein (10 ppm final concentrationin reaction mixture) of one of the following lipases (Novozymes) wereadded to one of each flask: Lipolase® 100 L, Lipex® 100L, Lipoclean®2000T, Lipozyme® CALB L, Novozyme® CALA L, and Palatase 20000L; nolipase was added to the seventh flask. The resulting mixtures werestirred at 55° C. in stoppered flasks, and 3-mL samples were withdrawnfrom each reaction mixture at 1 h, 2 h, 4 h and 6 h and immediatelyfrozen in dry ice until analyzed for wt % lipid (derivatized as fattyacid methyl esters, FAME) and for wt % free fatty acid (FFA, derivatizedas fatty acid methyl esters, FAME) according to the method described byReference 1, and the percent free fatty acid content was calculatedrelative to the total combined concentrations of lipid and free fattyacid was determined for each sample. Results are shown in Table 9.

TABLE 9 Percent free fatty acid content (% FFA) of a mixture of groundwhole corn kernels using lipase treatment at 55° C. prior toliquefaction % FFA time 0 h 1 h 2 h 4 h 6 h Lipolase ® 100L 33 56 74 7679 Lipex ® 100L 34 66 81 83 83 Lipoclean ® 2000T 38 55 73 69 65Lipozyme ® CALB L 39 38 37 43 41 Novozyme ® CALA L 37 40 44 44 45Palatase ® 20000L 37 49 59 62 66 no enzyme 38 33 37 41 42

Example 26 Lipase Treatment of Ground Whole Corn Kernels Prior toSimultaneous Saccharification and Fermentation with In-Situ ProductRemoval Using Oleyl Alcohol

Three fermentations were run as described above in Examples 7, 8, and10. For fermentations run as described in Examples 7 and 10, lipase (10ppm of Lipolase® total soluble protein) was added to the suspension ofground corn and heated at 55° C. for 6 h prior to Liquefaction toproduce a liquefied corn mash containing heat-inactivated lipase. Nolipase was added to the suspension of ground corn used to prepareliquefied corn mash for the fermentation described in Example 8, but thesuspension was subjected to the same heating step at 55° C. prior toliquefaction. The % FFA in lipase-treated liquefied corn mash preparedfor fermentations run as described in Examples 7 and 10 was 83% and 86%,respectively, compare to 41% without lipase treatment (Example 8). Overthe course of the fermentations, the concentration of FFA did notdecrease in any of the fermentations, including that containingheat-inactivated lipase. The % FFA in the OA phase of the fermentationrun according to Examples 7 and 10 (with heat inactivation of lipaseprior to fermentation) was 97% at 70 h (end of run (EOR)), compared toonly 49% FFA for the fermentation run according to Example 8 whereground whole corn kernels had not been treated with lipase prior toliquefaction. Results are shown in Table 10.

TABLE 10 Lipid and free fatty acid content of fermentations containingoleyl alcohol as ISPR solvent and heat-inactivated lipase (lipasetreatment of ground corn suspension prior to liquefaction) lipids FFAlipids FFA lipid + fermentation lipase time (h), sample (wt %) (wt %)(g) (g) FFA (g) % FFA Example 7 10 ppm pre-lipase/pre-liq. 0.65 0.22 7.12.4 9.4 25 Example 7 10 ppm post-lipase/pre-liq. 0.22 0.65 2.4 7.0 9.574 Example 7 10 ppm liquefied mash 0.17 0.79 1.8 8.5 10.3 83 Example 710 ppm 0.3 h, broth 0.16 0.79 1.8 8.9 10.7 83 Example 7 10 ppm 4.8 h,broth 0.14 0.31 1.6 3.5 5.1 69 Example 7 10 ppm 4.8 h, OA 0.04 0.68 0.35.4 5.6 95 Example 7 10 ppm  29 h, broth 0.10 0.12 1.2 1.3 2.5 53Example 7 10 ppm  29 h, OA 0.03 1.05 0.2 8.2 8.4 98 Example 7 10 ppm  53h, broth Example 7 10 ppm  53 h, OA 0.07 1.14 0.5 9.0 9.5 95 Example 710 ppm  70 h, broth 0.11 0.07 1.2 0.8 2.0 39 Example 7 10 ppm  70 h, OA0.03 1.10 0.2 8.7 8.9 97 Example 8 none pre-lipase/pre-liq. 0.62 0.236.7 2.5 9.2 27 Example 8 none post-lipase/pre-liq. 0.57 0.26 6.2 2.8 9.031 Example 8 none liquefied mash 0.52 0.36 5.6 4.0 9.6 41 Example 8 none0.3 h, broth 0.50 0.33 5.7 3.8 9.4 40 Example 8 none 4.8 h, broth 0.470.14 5.3 1.6 6.9 24 Example 8 none 4.8 h, OA 0.12 0.32 1.0 2.9 3.9 73Example 8 none  29 h, broth 0.30 0.05 3.4 0.6 4.0 16 Example 8 none  29h, OA 0.31 0.46 2.7 4.1 6.9 60 Example 8 none  53 h, broth Example 8none  53 h, OA 0.47 0.50 4.2 4.4 8.6 51 Example 8 none  70 h, broth 0.220.04 2.5 0.5 3.0 17 Example 8 none  70 h, OA 0.40 0.39 3.6 3.5 7.0 49Example 10 10 ppm pre-lipase/pre-liq. 0.67 0.23 7.4 2.5 9.9 25 Example10 10 ppm post-lipase/pre-liq. 0.19 0.69 2.1 7.6 9.7 78 Example 10 10ppm liquefied mash 0.14 0.85 1.6 9.4 11.0 86 Example 10 10 ppm 0.3 h,broth 0.13 0.82 1.5 9.4 10.9 86 Example 10 10 ppm 4.8 h, broth 0.11 0.291.3 3.3 4.6 72 Example 10 10 ppm 4.8 h, OA 0.04 0.60 0.3 5.2 5.6 94Example 10 10 ppm  29 h, broth 0.09 0.14 1.0 1.6 2.6 61 Example 10 10ppm  29 h, OA 0.01 0.96 0.1 8.4 8.5 99 Example 10 10 ppm  53 h, brothExample 10 10 ppm  53 h, OA 0.02 0.95 0.2 8.3 8.4 98 Example 10 10 ppm 70 h, broth 0.09 0.08 1.1 0.9 1.9 45 Example 10 10 ppm  70 h, OA 0.030.99 0.3 8.7 9.0 97

Example 27 Lipase Treatment of Ground Whole Corn Kernels or LiquefiedCorn Mash for Simultaneous Saccharification and Fermentation withIn-Situ Product Removal Using Corn Oil Fatty Acids (COFA)

Five fermentations were run as described above in Examples 9, 11, 12,13, and 14. For the fermentations run as described in Examples 9, 13,and 14, lipase (10 ppm of Lipolase® total soluble protein) was addedafter Liquefaction and there was no heat-inactivation of lipase.Fermentations run as described in Examples 9 and 14 had 5 g/L of ethanoladded prior to inoculation, whereas the fermentation run as described inExample 13 had no added ethanol. The fermentations run as described inExamples 11 and 12 employed the addition of 10 ppm Lipolase® totalsoluble protein to the suspension of ground corn prior to liquefaction,resulting in heat inactivation of lipase during liquefaction. Thefermentation run as described in Example 11 had 5 g/L of ethanol addedprior to inoculation, whereas the fermentation run as described inExample 12 had no added ethanol. The final total grams of isobutanol(i-BuOH) present in the COFA phase of the fermentations containingactive lipase was significantly greater than the final total grams ofi-BuOH present in the COFA phase of the fermentations containinginactive lipase. The final total grams of isobutanol (i-BuOH) present inthe fermentation broths containing active lipase were only slightly lessthan the final total grams of i-BuOH present in the fermentation brothscontaining inactive lipase, such that the overall production of i-BuOH(as a combination of free i-BuOH and isobutyl esters of COFA (FABE)) wassignificantly greater in the presence of active lipase when compared tothat obtained in the presence of heat-inactivated lipase. Results areshown in Tables 11 and 12.

TABLE 11 Dependence of the production of free isobutanol (i-BuOH) andisobutyl esters of COFA (FABE) in fermentations containing corn oilfatty acids (COFA) as ISPR solvent on presence (Examples 9, 13, and 14)or absence (Examples 11 and 12) of active lipase (COFA phase analysis)fermen- g i-BuOH total g tation g i-BuOH/ g FABE/ from FABE/ i-BuOH/fermentation time (h) kg COFA kg COFA kg COFA kg COFA Example 9 4.5 2.40.0 0 2.4 Example 9 28.8 5.4 70.9 16.5 22.0 Example 9 52.4 8.9 199.046.4 55.3 Example 9 69.3 4.9 230.9 53.9 69.3 Example 11 6.6 2.3 0.0 0.02.3 Example 11 53.5 25.1 2.9 0.6 25.7 Example 11 71.1 24.4 6.3 1.4 25.8Example 12 6.6 2.3 0.0 0.0 2.3 Example 12 53.5 12.8 1.6 0.4 13.2 Example12 71.1 12.8 3.0 0.7 13.5 Example 13 6.6 2.3 0.0 0.0 2.3 Example 13 53.54.9 72.1 16.0 20.9 Example 13 71.1 4.6 91.4 20.3 24.9 Example 14 6.6 2.10.0 0.0 2.1 Example 14 53.5 9.8 197.2 43.8 53.6 Example 14 71.1 4.9244.5 54.3 59.2

TABLE 12 Dependence of the production of free isobutanol (i-BuOH) andisobutyl esters of COFA (FABE) in fermentations containing corn oilfatty acids (COFA) as ISPR solvent on presence (Examples 9, 13, and 14)or absence (Examples 11 and 12) of active lipase (fermentation brothanalysis) fermen- g i-BuOH total g tation g i-BuOH/ g FABE/ from FABE/i-BuOH/ sample time (h) kg broth kg broth kg broth kg broth Example 94.5 0.0 0.0 0 0 Example 9 28.8 0.0 12.6 2.9 2.9 Example 9 52.4 0.0 30.37.1 7.1 Example 9 69.3 0.0 24.7 5.8 5.8 Example 11 6.6 0.0 0.0 0 0.0Example 11 53.5 9.8 0.0 0 9.8 Example 11 71.1 9.5 0.0 0 9.5 Example 126.6 0.0 0.0 0 0 Example 12 53.5 3.8 0.0 0.0 3.8 Example 12 71.1 5.1 0.00.0 5.1 Example 13 6.6 0.0 0.0 0 0 Example 13 53.5 2.1 3.0 0.7 2.8Example 13 71.1 2.1 7.4 1.6 3.7 Example 14 6.6 0.0 0.0 0 0.0 Example 1453.5 2.9 22.4 5.0 7.9 Example 14 71.1 3.3 19.3 4.3 7.6

Example 28 Dependence of Isobutyl-COFA Ester Concentration onAqueous/COFA Ratio in Lipase-Catalyzed Reactions

Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic acidbuffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), lipase(Lipolase® 100 L; Novozymes) and corn oil fatty acids prepared from cornoil (Table 13) were stirred at 30° C., and samples were withdrawn fromeach reaction mixture at predetermined times, immediately centrifuged,and the aqueous and organic layers separated and analyzed for isobutanol(i-BuOH) and isobutyl esters of corn oil fatty acids (i-BuO-COFA) (Table14).

TABLE 13 Reaction conditions for conversion of isobutanol (i-BuOH) toisobutyl esters of corn oil fatty acids (i-BuO-COFA) MES (0.2M) i-BuOHCOFA lipase reaction (g) (g) (g) (ppm) 1 45.96 3.6 43.4 10 2 45.96 3.621.7 10 3 45.96 3.6 10.85 10 4 45.96 3.6 43.4 4 5 45.96 3.6 43.4 0

TABLE 14 Weights of isobutanol (i-BuOH) and isobutyl esters of corn oilfatty acids (i-BuO-COFA) present in the aqueous fraction (AQ) andorganic fraction (ORG) for reactions described in Table 13 i-BuOH fromtotal total free i-BuO- i-BuO- i-BuOH i-BuOH i-BuOH COFA COFA (g) (g)(g) (g) (g) reaction time (h) (AQ) (ORG) (ORG) (ORG) (ORG) 1 0.1 0.772.83 2.77 0.05 0.24 1 1 0.76 2.84 2.58 0.25 1.13 1 2 0.74 2.86 2.41 0.442.00 1 4 0.66 2.94 2.05 0.89 4.03 1 6 0.63 2.97 1.43 1.54 6.93 1 21.50.28 3.32 0.34 2.98 13.4 1 25.5 0.23 3.37 0.29 3.08 13.8 2 0.1 1.17 2.432.36 0.07 0.30 2 1 1.09 2.51 2.26 0.24 1.10 2 2 1.07 2.53 2.19 0.34 1.522 4 1.03 2.57 1.99 0.59 2.64 2 6 1.00 2.60 1.70 0.90 4.04 2 21.5 0.752.85 0.58 2.27 10.2 2 25.5 0.59 3.01 0.49 2.52 11.4 3 0.1 1.56 2.04 1.980.06 0.27 3 1 1.55 2.05 1.77 0.28 1.24 3 2 1.49 2.11 1.65 0.46 2.08 3 41.45 2.15 1.28 0.87 3.92 3 6 1.33 2.27 0.96 1.31 5.92 3 21.5 1.12 2.480.26 2.22 10.0 3 25.5 0.88 2.72 0.26 2.46 11.1 4 0.1 0.84 2.76 2.75 0.020.07 4 1 0.78 2.82 2.73 0.09 0.40 4 2 0.83 2.77 2.59 0.17 0.79 4 4 0.782.82 2.44 0.38 1.71 4 6 0.78 2.82 2.10 0.72 3.25 4 21.5 0.58 3.02 1.121.90 8.57 4 25.5 0.51 3.09 0.97 2.11 9.51 5 0.1 0.90 2.70 2.70 0.00 0.005 1 0.90 2.70 2.70 0.00 0.00 5 2 0.92 2.68 2.68 0.00 0.00 5 4 0.89 2.712.70 0.00 0.02 5 6 0.92 2.68 2.62 0.06 0.29 5 21.5 0.90 2.70 2.62 0.080.37 5 25.5 0.89 2.71 2.62 0.09 0.41

Example 29 Dependence of Isobutyl-COFA Ester Concentration onAqueous/COFA Ratio in Lipase-Catalyzed Reactions

Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic acidbuffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol) or n-butanol,lipase (Lipolase® 100 L; Novozymes) and corn oil fatty acids preparedfrom corn oil (Table 15) were stirred at 30° C., and samples werewithdrawn from each reaction mixture at predetermined times, immediatelycentrifuged, and the aqueous and organic layers separated and analyzedfor isobutanol (i-BuOH) or n-butanol (n-BuOH) and isobutyl- or butylesters of corn oil fatty acids (BuO-COFA) (Table 16).

TABLE 15 Reaction conditions for conversion of isobutanol (i-BuOH) or n-butanol (n-BuOH) to butyl esters of corn oil fatty acids (BuO-COFA) MES(0.2M) butanol COFA lipase reaction butanol (g) (g) (g) (ppm) 6iso-butanol 45.96 3.6 13.5 10 7 n-butanol 45.96 3.6 13.5 10 8iso-butanol 45.96 3.6 13.5 0 9 isobutanol 45.96 3.6 13.5 4

TABLE 16 Weights of isobutanol (i-BuOH) or n-butanol (n-BuOH) and butylesters of corn oil fatty acids (BuO-COFA) present in the aqueousfraction (AQ) and organic fraction (ORG) for reactions described inTable 15 i-BuOH total i- from i-BuO- total i-BuOH total i-BuOH BuOHi-BuOH COFA reaction time (h) (g) (AQ) (g) (ORG) (g) (ORG) (g) (ORG) (g)(ORG) 6 0 1.46 2.14 2.11 0.04 0.16 6 2 1.41 2.19 1.63 0.56 2.51 6 4 1.272.33 1.31 1.02 4.58 6 21 0.66 2.94 0.29 2.65 12.0 6 25 0.60 3.00 0.262.73 12.3 6 46 0.54 3.06 0.22 2.83 12.8 n-BuOH from n- n-BuO- totaln-BuOH total n-BuOH n-BuOH BuO-COFA COFA (g) (AQ) (g) (ORG) (g) (ORG)(g) (ORG) (g) (ORG) 7 0 1.31 2.29 2.26 0.03 0.11 7 2 1.26 2.34 1.89 0.452.03 7 4 1.20 2.40 1.66 0.74 3.35 7 21 0.81 2.79 0.50 2.29 10.3 7 250.77 2.83 0.40 2.43 11.0 7 46 0.50 3.10 0.23 2.87 12.9 i-BuOH from i-i-BuO- total i-BuOH total i-BuOH i-BuOH BuO-COFA COFA (g) (AQ) (g) (ORG)(g) (ORG) (g) (ORG) (g) (ORG) 8 0 1.62 1.98 1.98 0.00 0.01 8 2 1.56 2.042.04 0.00 0.00 8 4 1.59 2.01 2.01 0.00 0.00 8 21 1.59 2.01 2.00 0.010.04 8 25 1.55 2.05 2.04 0.01 0.04 8 46 1.45 2.15 2.12 0.02 0.11 i-BuOHfrom i- i-BuO- total i-BuOH total i-BuOH i-BuOH BuO-COFA COFA (g) (AQ)(g) (ORG) (g) (ORG) (g) (ORG) (g) (ORG) 9 0 1.57 2.03 2.02 0.01 0.04 9 21.54 2.06 1.86 0.19 0.86 9 4 1.44 2.16 1.79 0.36 1.64 9 21 1.14 2.460.95 1.51 6.82 9 25 1.10 2.50 0.83 1.67 7.50 9 46 0.78 2.82 0.44 2.3710.7

Example 30 Production of Iso-Butyl Oleate by Lipase-Catalyzed Reactionof Iso-Butanol and Oleic Acid

Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic acidbuffer (0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), lipase (0 ppmor 10 ppm Lipolase® 100 L; Novozymes) and oleic acid (Alfa Aesar) (Table17) were stirred at 30° C., and samples were withdrawn from eachreaction mixture at predetermined times, immediately centrifuged, andthe aqueous and organic layers separated and analyzed for isobutanol(i-BuOH) and iso-butyl oleate (i-BuO-oleate) (Table 18).

TABLE 17 Reaction conditions for conversion of isobutanol (i-BuOH) toiso-butyl oleate (i-BuO-oleate) MES (0.2M) i-BuOH oleic acid lipasereaction (g) (g) (g) (ppm) 10 46.11 3.64 14.62 10 11 46.10 3.59 14.40 0

TABLE 18 Weights of isobutanol (i-BuOH) and iso-butyl oleate(i-BuO-COFA) present in the aqueous fraction (AQ) and organic fraction(ORG) for reactions described in Table 17. i-BuOH from total totali-BuO- i-BuO- i-BuOH i-BuOH i-BuOH oleate oleate (g) (g) (g) (g) (g)reaction time (h) (AQ) (ORG) (ORG) (ORG) (ORG) 10 0 1.37 2.28 2.24 0.040.18 10 2 1.30 2.34 1.95 0.40 1.81 10 4 1.28 2.37 1.82 0.55 2.53 10 61.22 2.42 1.71 0.72 3.27 10 23 0.92 2.72 0.71 2.01 9.20 10 27 0.89 2.750.65 2.11 9.62 10 47 0.81 2.84 0.55 2.29 10.5 10 51 0.82 2.83 0.54 2.2910.5 11 0 1.44 2.16 2.16 0.00 0.00 11 2 1.45 2.15 2.15 0.00 0.00 11 41.44 2.16 2.16 0.00 0.00 11 6 1.43 2.16 2.16 0.00 0.00 11 23 1.49 2.102.10 0.01 0.02 11 27 1.46 2.14 2.13 0.01 0.04 11 47 1.48 2.12 2.09 0.020.10 11 51 1.52 2.07 2.05 0.02 0.11

Example 31 Production of Iso-Butyl Oleate by Lipase-Catalyzed Reactionof Iso-Butanol and Oleic Acid

Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic acidbuffer (MES, 0.20 M, pH 5.2), isobutanol (2-methyl-1-propanol), oleicacid (Alfa Aesar), and lipase (10 ppm) from Lipolase® 100L, Lipex® 100L,Lipozyme® CALB L, Novozyme® CALA L, Palatase® from Novozymes, or lipase(10 ppm) from Pseudomonas fluorescens, Pseudomonas cepacia, Mucormiehei, hog pancreas, Candida cylindracea, Rhizopus niveus, Candidaantarctica, Rhizopus arrhizus or Aspergillus from SigmaAldrich (Table19), were stirred at 30° C., and samples were withdrawn from eachreaction mixture at predetermined times, immediately centrifuged, andthe aqueous and organic layers separated and analyzed for isobutanol(i-BuOH) and iso-butyl oleate (i-BuO-oleate) (Table 20).

TABLE 19 Reaction conditions for conversion of isobutanol (i-BuOH) toiso-butyl oleate (i-BuO-oleate) MES (0.2M) i-BuOH oleic acid lipase (g)(g) (g) (ppm) 46.105 3.601 13.72 10

TABLE 20 Weights of isobutanol (i-BuOH) and iso-butyl oleate(i-BuO-oleate) present in the aqueous fraction (AQ) and organic fraction(ORG) for reactions described in Table 19 total i- total i- i-BuOH fromi-BuO- BuOH BuOH i-BuOH i-BuO-oleate oleate lipase time (h) (g) (AQ) (g)(ORG) (g) (ORG) (g) (ORG) (g) (ORG) Lipolase ® 100L 23 1.55 2.05 1.470.59 2.68 Lipex ® 100L 23 0.65 2.95 0.30 2.65 12.09 Lipozyme ® CALB L 231.01 2.59 0.82 1.77 8.08 Novozyme ® CALA L 23 1.39 2.22 2.16 0.06 0.27Palatase ® 23 1.27 2.33 1.43 0.91 4.14 Pseudomonas fluorescens 23 1.382.22 1.97 0.25 1.14 Pseudomonas cepacia 23 1.39 2.21 1.95 0.26 1.20Mucor miehei 23 1.29 2.31 1.57 0.75 3.42 hog pancreas 23 1.40 2.20 2.190.01 0.04 Candida cylindracea 23 1.15 2.45 1.08 1.37 6.25 Rhizopusniveus 23 1.39 2.21 2.19 0.02 0.11 Candida antarctica 23 1.37 2.24 2.080.15 0.69 Rhizopus arrhizus 23 1.01 2.59 0.81 1.78 8.12 Aspergillus 231.36 2.24 2.06 0.18 0.82

Example 32 Production of Iso-Butyl COFA Esters byPhospholipase-Catalyzed Reaction of Iso-Butanol and Corn Oil Fatty Acids(COFA)

Reaction mixtures containing aqueous 2-(N-morpholino)ethanesulfonic acidbuffer (0.20 M, pH 5.3), isobutanol (2-methyl-1-propanol), phospholipase(Phospholipase A; SigmaAldrich, L3295-250) and corn oil fatty acidsprepared from corn oil were stirred at 30° C. (Table 21), and sampleswere withdrawn from each reaction mixture at predetermined times,immediately centrifuged, and the aqueous and organic layers separatedand analyzed for isobutanol (i-BuOH) and isobutyl esters of corn oilfatty acids (i-BuO-COFA) (Table 22).

TABLE 21 Reaction conditions for conversion of isobutanol (i-BuOH) toisobutyl esters of corn oil fatty acids (i-BuO-COFA) MES buffer i-BuOHCOFA lipase reaction # (0.2M) (g) (g) (g) (ppm) 1 46.1 3.6 14.7 10 246.1 3.6 14.7 3 3 46.1 3.6 14.7 0

TABLE 22 Weights of isobutanol (i-BuOH) and isobutyl esters of corn oilfatty acids (i-BuO-COFA) present in the aqueous fraction (AQ) andorganic fraction (ORG) for reactions described in Table 21 i-BuOH fromtotal total free i-BuO- i-BuO- i-BuOH i-BuOH i-BuOH COFA COFA (g) (g)(g) (g) (g) reaction time (h) (AQ) (ORG) (ORG) (ORG) (ORG) 1 0.1 1.292.39 2.39 0.00 0.00 1 2 1.24 2.44 2.38 0.06 0.26 1 20 1.25 2.43 2.220.21 0.96 1 24 1.26 2.42 2.19 0.23 1.03 1 44 1.27 2.41 2.13 0.28 1.28 148 1.22 2.46 2.15 0.31 1.41 2 0.1 1.27 2.34 2.34 0.00 0.00 2 2 1.25 2.352.33 0.02 0.08 2 20 1.24 2.37 2.30 0.07 0.30 2 24 1.22 2.38 2.31 0.070.32 2 44 1.33 2.28 2.18 0.10 0.44 2 48 1.23 2.38 2.27 0.11 0.48 3 0.11.27 2.33 2.33 0.00 0.00 3 2 1.26 2.34 2.34 0.00 0.00 3 20 1.22 2.382.37 0.01 0.07 3 24 1.25 2.35 2.33 0.02 0.08 3 44 1.24 2.36 2.32 0.040.18 3 48 1.24 2.36 2.32 0.04 0.18

Example 33 Comparison of Partition Coefficients for Isobutanol BetweenWater and Extractant

Aqueous solutions of isobutanol (30 g/L) were mixed with corn oil fattyacids (COFA), oleic acid, or corn oil triglycerides, and their measuredpartition coefficients reported in the table relative to the measuredpartition coefficient for oleyl alcohol. Results are shown in Table 23.

TABLE 23 Relative partition coefficients for isobutanol (30 g/L) betweenwater and extractant isobutanol partition coefficient, extractantrelative to oleyl alcohol oleyl alcohol 100% corn oil fatty acids 91%corn oil fatty acid isobutyl esters 43% corn oil triglycerides 10%

Example 34 Hydroxylated Triglycerides from Corn Oil

To a three-neck 500 mL flask equipped with a mechanical stirrer andaddition funnel was added corn oil (50.0 g), toluene (25.0 mL),Amberlyte IR-120 resin (12.5 g), and glacial acetic acid (7.5 g). Theresulting mixture was heated to 60° C., and then hydrogen peroxide (41.8g of 30% H₂O₂ in water) was added dropwise over one hour. The mixturewas stirred at 60° C. for two hours, upon which time the reactionmixture was worked up: resin was removed by filtration, and the filtratepartitioned between ethyl acetate (75 mL) and water (50 mL). After thelayers were separated, the organic layer was washed with sat. aq. NaHCO₃solution (50 mL), and brine (50 mL). The organic layer was dried overanh. Na₂SO₄ and concentrated in vacuo to obtain 48.9 g of yellow oil.The ¹H NMR analysis of the crude reaction product showed that 63% ofdouble bonds were epoxidized.

A. Corn Oil Hydroxylation (63% Hydroxylation)

To a three-neck 500 mL flask equipped with a mechanical stirrer andaddition funnel was added corn oil (50.0 g), toluene (25.0 mL),Amberlyte IR-120 resin (12.5 g), and glacial acetic acid (7.5 g). Theresulting mixture was heated to 60° C., and then hydrogen peroxide (41.8g of 30% H₂O₂ in water) was added dropwise over one hour. The mixturewas stirred at 60° C. for two hours, upon which time the reactionmixture was worked up: resin was removed by filtration, and the filtratepartitioned between ethyl acetate (75 mL) and water (50 mL). After thelayers were separated, the organic layer was washed with sat. aq. NaHCO₃solution (50 mL), and brine (50 mL). The organic layer was dried overanh. Na₂SO₄ and concentrated in vacuo to obtain 48.9 g of yellow oil.The ¹H NMR analysis of the crude reaction product showed that 63% ofdouble bonds were epoxidized.

To a 500 mL round bottom flask was added epoxidized corn oil (20.0 g),tetrahydrofuran (THF) (100.0 mL), and sulfuric acid (50 mL of 1.7 Maqueous solution). The cloudy mixture was stirred for two hours at 50°C., and then worked up by partitioning between water (100 mL) and ethylacetate (200 mL). The organic layer was washed with water (3×50 mL) andthen brine (50 mL). The organic layer was dried over anh. Na₂SO₄ andconcentrated in vacuo to obtain 19.9 g of dark yellow oil (63%hydroxylation corn oil).

B. Corn Oil Hydroxylation (47% Hydroxylation)

To a three-neck 500 mL flask, equipped with a mechanical stirrer andaddition funnel was added corn oil (50.0 g), toluene (25.0 mL),Amberlyte IR-120 resin (12.5 g), and glacial acetic acid (7.5 g). Theresulting mixture was heated to 60° C., and then hydrogen peroxide (41.8g of 30% H₂O₂ in water) was added dropwise over one hour. The mixturewas stirred at 60° C. for one hour, upon which time the reaction mixturewas worked up: the resin was removed by filtration, and the filtratepartitioned between ethyl acetate (75 mL) and water (50 mL). After thelayers were separated, the organic layer was washed with sat. aq. NaHCO₃solution (50 mL), and brine (50 mL). The organic layer was dried overanh. Na₂SO₄ and concentrated in vacuo to obtain 49.8 g of yellow oil.The ¹H NMR analysis of the crude reaction product showed that 47% ofdouble bonds were epoxidized.

To a 500 mL round bottom flask was added epoxidized corn oil (20.0 g),THF (100.0 mL), and sulfuric acid (50 mL of 1.7M aqueous solution). Thecloudy mixture was stirred for two hours at 50° C., and then worked upby partitioning between water (100 mL) and ethyl acetate (200 mL). Theorganic layer was washed with water (3×50 mL) and then brine (50 mL).The organic layer was dried over anh. Na₂SO₄ and concentrated in vacuoto obtain 19.2 g of dark yellow oil (47% hydroxylation corn oil).

C. Corn Oil Hydroxylation (28% Hydroxylation)

To a three-neck 500 mL flask, equipped with a mechanical stirrer andaddition funnel was added corn oil (50.0 g), toluene (25.0 mL),Amberlyte IR-120 resin (12.5 g), and glacial acetic acid (7.5 g). Theresulting mixture was heated to 60° C., and then hydrogen peroxide (41.8g of 30% H₂O₂ in water) was added dropwise over one hour. The mixturewas stirred at 60° C. for two hours, upon which time the reactionmixture was worked up: the resin was removed by filtration, and thefiltrate partitioned between ethyl acetate (75 mL) and water (50 mL).After the layers were separated, the organic layer was washed with sat.aq. NaHCO₃ solution (50 mL), and brine (50 mL). The organic layer wasdried over anh. Na₂SO₄ and concentrated in vacuo to obtain 47.2 g ofyellow oil. The ¹H NMR analysis of the crude reaction product showedthat 28% of double bonds were epoxidized.

To a 500 mL round bottom flask was added epoxidized corn oil (20.0 g),THF (100.0 mL), and sulfuric acid (50 mL of 1.7M aqueous solution). Thecloudy mixture was stirred for two hours at 50° C., and then worked upby partitioning between water (100 mL) and ethyl acetate (200 mL). Theorganic layer was washed with water (3×50 mL) and then brine (50 mL).The organic layer was dried over anh. Na₂SO₄ and concentrated in vacuoto obtain 20.3 g of dark yellow oil (28% hydroxylation corn oil).

Partition Coefficient Measurement

To a 5 mL vial was added 0.910 g of the 67% hydroxylated corn oil, and0.910 mL of 3 wt % i-BuOH water solution. The biphasic mixture wasvigorously stirred using Vortex Genie® for 10 minutes. Upon mixing, theseparation of layers was aided by centrifuging the mixture using FisherScientific Centrific 228 centrifuge (3300 rpm) for 10 minutes. 0.100 gof both layers were taken. The organic, upper layer was diluted to 1.00mL with toluene solution of ethylene glycol diethylether (10.1 mg/mL),and the water layer was diluted to 1.00 mL with methanol solution ofethylene glycol diethylether (10.2 mg/mL). The concentrations of i-BuOHin both phases were measured using a calibrated gas chromatograph (GC).The same procedure was repeated for 47% and 28% hydroxylated corn oil.The partition coefficient thus measured was 3.2 for the 67% hydroxylatedcorn oil, 2.3 for the 47% hydroxylated corn oil, and 2.1 for the 28%hydroxylated corn oil.

The above outlined procedure was repeated with 6% i-BuOH water solution.The partition coefficients for 67%-, 47%-, and 28%-hydroxylated cornoils were 2.9, 2.9, and 2.0, respectively.

Example 34 Fatty Amides Plus Fatty Acids, and Pure Fatty Amides fromCorn Oil

Corn oil was reacted with aqueous ammonium hydroxide in a manner similarto that described by Roe, et al., J. Am. Oil Chem. Soc. 29:18-22, 1952.Mazola® corn oil (0.818 L, 755 g) was placed in a 1 gallon stainlesssteel reactor to which was added 1.71 L (1540 g) of aqueous ammoniumhydroxide (28% as NH₃). The reactor was heated with stirring to 160° C.and was maintained at that temperature with stirring for 7 h duringwhich time the pressure reached 400 psi. The reactor was cooled and theproduct, a creamy white solid, was removed and the reactor rinsed withethyl acetate. The product was dissolved in 5 L ethyl acetate and washed5 times with 500 mL each of water which was neutralized with H₂SO₄. Theethyl acetate was then dried over anhydrous Na₂SO₄ and the solventremoved on a rotary evaporator leaving a light brown soft solid.

¹³C NMR in CDCl₃ indicated that the product contained an approximate 2:1ratio of fatty amide to fatty acid and that the conversion of the cornoil to product was quantitative. The product had a melting point of57-58° C., but dropped about 11° C. when saturated with water.

Pure corn oil fatty amide was synthesized from corn oil according toKohlhase, et al., J. Am. Oil Chem. Soc. 48:265-270, 1971 using anhydrousammonia with ammonium acetate as a catalyst.

Three grams of ammonium acetate were placed in a 400 mL stainless steelshaker tube to which was added 51.8 g of corn oil. Anhydrous ammonia(89.7 g) was then added and the reactor sealed and heated for 7 h at125° C. during which time the pressure reached 1300 psi. The reactor wascooled, the light colored solid removed and the reactor rinsed withethyl acetate. The product dissolved in ethyl acetate was then worked upas in the case of the fatty amide/fatty acid mixture above.

Fatty acids were synthesized from corn oil by base hydrolysis usingNaOH. Round bottom flask (5 L) was equipped with a mechanical stirrer,thermocouple, heating mantle, condenser, and nitrogen tee. Charged with500 g of food grade corn oil, 1 L of water and 75 g of sodium hydroxide.Mixture was heated to 90° C. and held for three hours, during which timeit became a single thick, emulsion-like single phase. At the end of thistime, TLC shows no remaining corn oil in the mixture. The mixture wasthen cooled to 72° C. and 500 mL of 25% sulfuric acid was added toacidify the mixture. It was then cooled to room temperature and 2 L ofdiethyl ether was added. The ether layer was washed 3×1 L with 1%sulfuric acid, 1×1 L with saturated brine, dried over MgSO₄, andfiltered. The ether was removed by rotovap and then the oil was purgedwith nitrogen overnight, obtaining 470 g of a yellow oil that partiallycrystallized overnight. Titration for free fatty acids via AOCS methodCa 5a-40 shows a fatty acid content of 95% expressed as oleic acid. Asample was silanized by reacting 104 mg with 100 uL ofN-methyl-N-(trimethylsilyl)trifluoroacetamide in 1 mL of dry pyridine.Gas chromatography-mass spectrometry (GCMS) analysis of the silanizedproduct shows the presence of the TMS derivatives of the 16:0, 18:2,18:1, 18:0, and 20:0 acids

Three preparations: (1) the 2:1 mixture of corn oil fatty amide and cornoil fatty acid from aqueous ammonia, (2) a 2:1 mixture of pure corn oilfatty amide:pure corn oil fatty acid, and (3) a 1:2 mixture of pure cornoil fatty amide:corn oil fatty acid, were all tested for their abilityto extract isobutanol from a 3% solution in water. Seven hundredmilligrams of each was added to 2.1 mL of water containing 3% isobutanolin a 20 mL scintillation vial and placed on a rotary shaker overnight at30° C. In all three cases, the organic phase became liquid at thistemperature, indicating a further lowering of the melting point with theuptake of isobutanol. Fifty microliters of the upper phase were dilutedwith either 200 μL of toluene containing ethylene glycol diethylether(10.068 mg/mL) as a GC standard or 200 μL of isopropanol containing thesame concentration of ethylene glycol diethylether. Fifty microliters ofthe lower phase was diluted with 150 μL of methanol and 50 μL ofisopropanol containing the same concentration of ethylene glycoldiethylether. The concentrations of isobutanol in both phases weredetermined using a calibrated GC. The partition coefficients measuredwere as follows: 3.81 for (1), 4.31 for (2), and 3.58 for (3).

Fatty amide/fatty acid aqueous ammonia preparation (1), and apreparation (1a) constituted by preparation (1) mixed 1:1 with pure cornoil fatty acid (equivalent to 1:2 fatty amide:fatty acid) were incubatedin shake flasks with fermentation broth containing the Saccharomycesbutanologen NGCI-070 at a ratio of 3 parts broth to 1 part amide/acidmixture. Preparation (1) was a soft solid, while preparation (1a) was aliquid at 30° C. Starting at a glucose concentration of 8.35 g/L, theshake flasks were then incubated for 25 h on an incubator shaker and theconsumption of glucose followed as a function of time. Table 24indicates that the fatty amide/fatty acid mixtures at both ratios werenot toxic to the butanologen and even showed higher rates of glucoseuptake than with oleyl alcohol.

TABLE 24 Glucose conc. (g/L) Flask Time = 0 18 hrs 25 hrs Oleyl Alcohol8.35 4.26 0 Oleyl Alcohol 8.35 4.46 0 2:1 Synthesized Fatty Amide:Fatty8.35 3.06 0 Acid Mix (Preparation (1)) 2:1 Synthesized Fatty Amide:Fatty8.35 3.22 0 Acid Mix (Preparation (1)) 1:1 Synthesized Fatty Amide Fatty8.35 2.73 0 Acid Mix:Pure Fatty Acids (Preparation (1a)) 1:1 SynthesizedFatty Amide Fatty 8.35 2.73 0 Acid Mix:Pure Fatty Acids (Preparation(1a))

Example 35 Fatty Alcohols from Corn Oil

With reference to the reaction of Equation IV above for producing fattyalcohols from corn oil, a 22L, round-bottom flask equipped with amechanical stirrer, reflux condenser with N₂ source, addition funnel,internal thermocouple, and rubber septum was flame-dried under nitrogen.The flask was charged with 132 g (3.30 moles) of 95% lithium aluminumhydride powder that is weighed out in a dry box and loaded into a solidsaddition funnel. The 22L flask was cooled with an ice bath, and 9.0liters of anhydrous THF were added into the reactor via a cannula. Theresulting slurry was cooled to 0-5° C. and a solution of 956 g (1.10moles) of Wesson® corn oil in 1.00 liter of anhydrous THF was addeddropwise over 2-3 hours while holding the reaction temperature at 5-20°C. After adding the corn oil, the slurry was stirred overnight atambient temperature. When the reaction was done, as verified by TLCchromatography, it was quenched by the dropwise addition of a solutionof 130 g of water dissolved in 370 mL of THF. Then 130 g of 15% aqueousNaOH solution was added followed by the addition of 400 g of water. Themixture was vigorously stirred while warming to room temperature andproduced a white granular solid. The solids were filtered off using afritted-glass filter funnel and washed with additional THF. The THF wasremoved on a rotary evaporator and the residue was taken up in 3.00liters of ethyl acetate. The product solution was washed with 2×1.00 Lof water, 1×1.00 L of brine, dried over Na₂SO₄, filtered, andconcentrated in vacuo to give 836 g (97%) of fatty alcohols as yellowoil. The crude fatty alcohol mixture was then distilled (140° C./1mmHg), and used in the following partition coefficients experiments.

Partition Coefficient Experiments

To each of the five 5-mL vials were added 1 mL of fatty alcohol mixture,and 1 mL of 3 wt % i-BuOH water solution. The biphasic mixture wasvigorously stirred using Vortex Genie® for 10, 20, 30, 40, and 60minutes, respectively. Upon mixing, the separation of layers was aidedby centrifuging the mixture using Fisher Scientific Centrific 228centrifuge (3300 rpm) for 10 minutes. 0.100 mL of both layers weretaken. The organic, upper layer was diluted to 1.00 mL with toluenesolution of ethylene glycol diethylether, and the water layer wasdiluted to 1.00 mL with methanol solution of ethylene glycoldiethylether. The concentrations of i-BuOH in both phases were measuredusing a calibrated GC. The partition coefficient thus measured was 2.70.

The same partition coefficient measurement, as described above was runfor 6 wt % i-BuOH concentration. The partition coefficient thus measuredwas 3.06.

While various embodiments of the present invention have been describedabove, it should be understood that they have been presented by way ofexample only, and not limitation. It will be apparent to persons skilledin the relevant art that various changes in form and detail can be madetherein without departing from the spirit and scope of the invention.Thus, the breadth and scope of the present invention should not belimited by any of the above-described exemplary embodiments, but shouldbe defined only in accordance with the following claims and theirequivalents.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method for producing an alcohol comprising: (a)providing biomass comprising water, fermentable sugars, and oil; (b)liquefying the biomass to produce a liquefied biomass; (c) contactingthe liquefied biomass with one or more catalysts whereby at least aportion of the oil is hydrolyzed to form an extractant, wherein apartition coefficient of the extractant for the product alcohol isgreater than a partition coefficient of the oil of the biomass for theproduct alcohol; (d) contacting the liquefied biomass with asaccharification enzyme capable of converting oligosaccharides intofermentable sugar; contacting the liquefied biomass with a fermentationbroth in a fermentation vessel; (e) fermenting the carbon source of theliquefied biomass to produce a product alcohol; (f) removing in situ theproduct alcohol from the fermentation broth by contacting the broth withthe extractant; and optionally steps (c) and (d) occur concurrently. 2.The method of claim 1, wherein the biomass comprises corn grain, wheat,rye, barley, soy, and mixtures thereof.
 3. The method of claim 1,wherein the extractant comprises fatty acids, fatty amides, fattyalcohols, fatty esters, or mixtures thereof.
 4. The method of claim 1,wherein the one or more catalysts is selected from esterase, lipase,phospholipase, and lysophospholipase.
 5. The method of claim 1, furthercomprising: inactivating the catalyst after at least a portion of theoil is hydrolyzed to form the extractant.
 6. The method of claim 1,wherein the product alcohol is butanol.
 7. The method of claim 1,wherein the biomass comprises hay, sorghum, switchgrass, and mixturesthereof.
 8. The method of claim 1, wherein the biomass comprisescellulosic material, lignocellulosic material, and mixtures thereof. 9.The method of claim 1, wherein the biomass comprises sugar cane bagasse.10. The method of claim 1, wherein the biomass comprises wheat straw,barley straw, rice straw, and mixtures thereof.
 11. The method of claim1, wherein the biomass comprises corn cobs, corn husks, corn stover, andmixtures thereof.
 12. The method of claim 1, wherein the biomasscomprises crop residues.